Luminogens for biological applications

ABSTRACT

A compound comprises a donor and an acceptor, wherein at least one donor (“D”) and at least one acceptor (“A”) may be arranged in an order of D-A; D-A-D; A-D-A; D-D-A-D-D; A-A-D-A-A; D-A-D-A-D; and A-D-A-D-A. The compound may be selected from the group consisting of: MTPE-TP, MTPE-TT, TPE-TP A-TT, PTZ-BT-TP A, NPB-TQ, TPE-TQ-A, MTPE-BTSe, DCDPP-2TP A, DCDPP-2TPA4M, DCDP-2TPA, DCDP-2TPA4M, TTS, ROpen-DTE-TPECM, and RClosed-DTE-TPECM. The compound may be used as a probe and may be functionalized with special targeted groups to image biological species. As non-limiting examples, the compound may be used in cellular cytoplasms or tissue imaging, blood vessel imaging, in vivo fluorescence imaging, brain vascular imaging, sentinel lymph node mapping, and tumor imaging, and the compound may be used as a photoacoustic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present patent application claims priority to provisional U.S.Patent Application No. 62/498,093 filed on Dec. 15, 2016; provisionalU.S. Patent Application No. 62/602,531 filed on Apr. 27, 2017; andprovisional U.S. Patent Application No. 62/605,977 filed on Sep. 6,2017, all of which were filed by the inventors hereof and areincorporated by reference herein in their entirety.

TECHNICAL FIELD

The present subject matter relates generally to organic chemistry,photophysics, and biology. In particular, the present subject matterrelates to a design strategy and application in developing luminogensand aggregation induced emission (AIE) luminogens (AIEgens) forbiological applications. In particular, the present subject matterrelates to red fluorescent AIE luminogen with high brightness, largeStokes shift, good biocompatibility, satisfactory photostability, andhigh two-photon absorption cross section.

BACKGROUND

Far red/near-infrared (FR/NIR) fluorescence-based technologies haveattracted considerable interest in recent years. FR/NIR imaging for invivo applications shows many advantages over imaging in the visibleregion, such as deep-tissue penetration due to diminished lightscattering, less photo-damage to the body due to low excitation energy,and high signal-to-noise ratio due to minimum interference from thebackground auto-fluorescence by biological substances in living systems.As a result, much effort has been devoted to developing FR/NIRfluorescent materials for biological applications.

A wide variety of nanostructured materials has been prepared and appliedfor biological imaging and therapy during the past decade, for example,carbonaceous nanomaterials and inorganic quantum dots (QDs). Althoughthese nanomaterials enjoy the advantage of strong light emission, theysuffer from some drawbacks, such as high toxicity and poorprocessability, which limit their further applications in the biologicalfield. Conventional organic dyes, on the other hand, possess goodbiocompatibility and processability, and several FR/NIR dyes have beensynthesized based on the unit of cyanine, BODIPY, rhodamine, andsquaraine. However, though all of these conventional FR/NIR dyes showbright emission in solution, their emission is partially or completelyquenched when aggregated in an aqueous medium or inside living cells.Thus, it is desirable to develop organic FR/NIR dyes with highfluorescent efficiency in the aggregate state for in vivo applications.

Unlike conventional organic dyes, aggregation-induced emissionluminogens (AIEgens) show stronger light emission in the aggregatestate. However, few FR/NIR AIEgens have been prepared because of thefollowing difficulties in synthesizing. To endow a luminogen with AIEproperties, one may introduce twisting units into the molecularstructure to hamper the π-π stacking. However, this will disrupt theπ-conjugation, leading to blue-shift, instead of red-shift in theemission. Therefore, a need exists for developing new AIEgens with longluminescence wavelengths to the FR/NIR spectral region and highbrightness for in vivobiological and diagnostic applications.

Further, red and near-infrared fluorescent biological imaging hasexhibited huge advantages in preclinical research and clinical practicefor noninvasive real-time tumor diagnosis and image-guided cancertherapy. Having light emission with a long wavelength plays anindispensable role in biological imaging because of several benefitsincluding deep tissue penetration, negligible biologicalauto-fluorescence in comparison to blue or green emission, lowphoto-damage caused by low excitation energy, high signal-to-noiseratio, etc. However, there are always difficulties associated with thedesign of red/near-infrared emissive dyes, especially with syntheticprocedures and less practicability for post-functionalization.

Recently, the fluorescent nanoparticles have caused tremendous interestfor biological imaging due to their high photo-stability, large specificsurface for post-modification, and intrinsic enhanced permeability andretention (EPR) effect for tumor tissue. However, the traditionalorganic fluorogens always possess a large planar conjugated structure.They can give strong emission in the solution state, but once fabricatedinto nanoparticles, the fluorescence is almost quenched. That is, theysuffer from the notorious aggregation-caused quenching (ACQ) effect,which is probably due to the formation of excimers upon aggregation andthus hampers their biological applications.

Carbon dots (CDs) are intrinsically emissive nanoparticles, but theiremission efficiency is rather low because of their unique emissivespecies. As such, it is difficult to design CDs with desired propertiesdue to their ambiguous luminescent mechanism. In contrast, for quantumdots (QDs), tunable emission with high luminescent efficiency is easy toachieve, but biological toxicity of inorganic QDs is unavoidable oncethey are employed for biological application. Further, the ACQ effectalso occurs when the concentration of QDs is increased.

In 2001, an abnormal photo-physical phenomenon of aggregation-inducedemission (AIE) was observed, which can thoroughly utilize aggregation toplay a positive role instead of a negative role in enhancingluminescence. The restriction of intramolecular motion (RIM) has beenproven theoretically and experimentally to be the cause of the AIEeffect, and with the aid of the RIM mechanism, plenty of organic AIEgenswith twisted conformation are tailored with colorful emissions as wellas rich functionalities.

To design luminogens with red/near-infrared emissions, the strategies ofincreasing molecular conjugation or introduction of electrondonor-acceptor (D-A) effect are always adopted. However, increasing themolecular conjugation may cause poor solubility of the luminogen andsimultaneously make it unstable. Thus, more attention has been directedto design of the red/near-infrared emitter by the rational introductionof the D-A effect. Designed red/near-infrared AIE luminogens (AIEgens)often incorporate tetraphenylethene (TPE) as molecular rotators becauseTPE is crucial for participating in dissipating the excitation stateenergy non-radiatively in the solution state and thus determining theAIE effect. However, introduction of TPE in the structure may causedifficulties in the synthesis, as the double bond in TPE easilyundergoes thermal-oxidation, photo-oxidation, or photo-isomerizationunder external stimuli, thus deteriorating the stability of the AIEgen.As such, a need exists for the preparation of red or near-infraredAIEgens with whole aromatic structures (without incorporating a TPEunit) which has simple synthetic steps as well as excellent stabilityfor further applications.

Furthermore, the fluorescence imaging technique is a powerfulvisualization method which enables real-time following and monitoring ofbiological processes in vivo. Blood vessels are the primary componentsfor the circulatory system. As such, the application of fluorescentprobes for the visualization of blood vasculature in vivo is of greatimportance, as it will shed light on better understanding, medicaldiagnosis, and therapeutics for diseases relevant to vascular leakageand obstruction, such as cerebral hemorrhage and cerebral thrombosis.

Two-photon fluorescence microscopy (TPM) has been widely utilized as animportant imaging tool for scientific research due to higher penetrationdepth with NIR excitation, higher spatial resolution with signal tonoise ratio, and less photobleaching. However, most reports are limitedfor utilization of emitters with a small two-photon absorption (2PA)cross section (δ_(max)<50 GM) or with a short wavelength emission.Because of this, some reports are restricted in cell imaging in vitro,without applications in vivo. Therefore, fluorescent materials withlonger wavelength emission, high quantum efficiency and high two-photonabsorption (2PA) cross section are in urgent demand.

To obtain a longer emission and/or a high two-photon absorption (2PA)cross section, traditional red molecule design has been based on theintroduction of nearly planar macrocyclic molecules with extendedπ-conjugation or with strong electron-donating and electron-acceptingunits. However, these designed molecules are prone to suffer fromaggregation-caused quenching (ACQ) effects in the aggregated state,which thus significantly weakens the performance of the traditional redemitters in relevant applications.

Since 2001, a phenomenon of aggregation-induced emission (AIE) has beenobserved in some organic luminophores free from the ACQ effect. Unlikeconventional luminophores, these luminophores are nonemissive in dilutesolutions but are induced to emit intensely when aggregated. Still now,a few red AIE emitters were reported. However, most of them show dualtwisted intramolecular charge transfer (TICT) and AIE effects withobvious background emission in THF solution and comparable emission inthe aggregated state. For example, the known compound TTB is emissive inTHF solution with only slight emission enhancement in the aggregatedstate. This raised the question as to how the molecule design of TTBcould be manipulated to suppress the background emission in solutionstate. Considering TICT and AIE effects are competitive effects, asimple and direct method is to enhance charge transfer (CT) property,which is an efficient nonradiative decay channel sensitive in solventpolarity in solution state. In this regard, a stronger D-A(donor-acceptor) system with two more arylamine units is introduced toTTB molecular structure for enhanced CT effect. Accordingly, moredevelopment has been desired in this area.

Fluorescence imaging is a powerful visualization method, which enablesreal-time following and monitoring of biological processes in vivo.Blood vessels are the primary components for the circulatory system. Assuch, there is a need for application of fluorescent probes for thevisualization of blood vasculature in vivo. Such an application is ofgreat importance, as it will shed light on better understanding, medicaldiagnosis, and therapeutics for the common diseases relevant to vascularleakage and obstruction, such as cerebral hemorrhage and cerebralthrombosis.

SUMMARY

In an embodiment, the present subject matter is directed to a compoundcomprising a donor and an acceptor, wherein each acceptor isindependently selected from the group consisting of:

wherein each D and D′ is the donor and is independently selected fromthe group consisting of:

wherein each X and X′ is independently selected from the groupconsisting of: O, S, Se, and Te;

wherein each R, R′, R″, R′″, and R″″ is independently selected from thegroup consisting of: F, H, alkyl, unsaturated alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, aminogroup, sulfonic group, alkylthio, alkoxy group, and

and wherein when any of R, R′, R″, R′″, and R″″ is a terminal functionalgroup, then each terminal functional group R, R′, R″, R′″, and R″″ isindependently selected from the group consisting of N₃, NCS, SH, NH₂,COOH, alkyne, N-Hydroxysuccinimide ester, maleimide, hydrazide, nitronegroup, —CHO, —OH, halide, and charged ionic group;

wherein each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ may be substitutedor unsubstituted and each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isindependently selected from the group consisting of H, alkyl,unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, C_(m)H_(2m+1), C₁₀H₇, C₁₂H₉, OC₆H₅, OC₁₀H₇, OC₁₂H₉,C_(m)H_(2m)COOH, C_(m)H_(2m)NCS, C_(m)H_(2m)N₃, C_(m)H_(2m)NH₂,C_(m)H_(2m)SO₃, C_(m)H_(2m)Cl, C_(m)H_(2m)Br, C_(m)H_(2m)I,

wherein n=0-2; and

wherein m=0 to 20.

In an embodiment, the compound exhibits aggregation-induced emission(AIE).

In an embodiment of the present subject matter, at least one donor andat least one acceptor are arranged in an order selected from the groupconsisting of:

-   -   Donor-Acceptor,    -   Donor-Acceptor-Donor,    -   Acceptor-Donor-Acceptor,    -   Donor-Donor-Acceptor-Donor-Donor,    -   Acceptor-Acceptor-Donor-Acceptor-Acceptor,    -   Donor-Acceptor-Donor-Acceptor-Donor, and    -   Acceptor-Donor-Acceptor-Donor-Acceptor.

In an embodiment, the present subject matter is directed to a probecomprising the present compound, wherein the probe is ared/near-infrared fluorescent probe.

In an embodiment, the present compound is functionalized with specialtargeted groups to image biological species.

In an embodiment, the present compound is fabricated in PEG/BSA and anyamphiphilic molecule matrix, wherein the probe works in a form ofnanoparticles. In an embodiment, the nanoparticles are incubated withcells or tissue and used for imaging cellular cytoplasms or tissue. Inan embodiment, the nanoparticles are injected into a blood vessel andused for blood vessel imaging.

In an embodiment, the structure is ROpen-DTE-TPECM and the presentcompound exhibits AIE characteristics and is used for in vivofluorescence imaging. In an embodiment, the structure isRClosed-DTE-TPECM and the present compound is used as a photoacousticagent.

In an embodiment, the present subject matter is directed to a probecomprising the present compound, wherein the probe is used for brainvascular imaging, sentinel lymph node mapping, and tumor imaging.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows mass spectrum of MTPE-TP.

FIG. 2 shows ¹H-NMR spectrum of MTPE-TP in CDCl₃.

FIG. 3 shows ¹³C-NMR spectrum of MTPE-TP in CDCl₃.

FIG. 4 shows absorption spectrum of MTPE-TP in DMSO.

FIG. 5 shows (A) photoluminescence (PL) spectra of MTPE-TP in DMSO andDMSO/water mixtures with increasing water fractions (fw) from 10% to90%. (B) Change in PL intensity of MTPE-TP versus water fraction inDMSO/water mixtures. Excitation at 530 nm.

FIG. 6 shows photoluminescence (PL) spectrum of MTPE-TP in the solidstate.

FIG. 7 shows photographs of (a) MTPE-TP in THF and THF/water mixtureswith 40% water fraction (fw), and (b) the solid sample taken under 365nm UV-light illumination.

FIG. 8 shows bright-field, fluorescent, and merged image of living HeLacells incubated with MTPE-TP dots for 12 h at 37° C. λ_(ex): 514 nm.

FIG. 9 shows mass spectrum of MTPE-TT.

FIG. 10 shows ¹H-NMR spectrum of MTPE-TT in CDCl₃.

FIG. 11 shows ¹³C-NMR spectrum of MTPE-TT in CDCl₃.

FIG. 12 shows absorption spectrum of MTPE-TT in DMSO.

FIG. 13 shows (A) photoluminescence (PL) spectra of MTPE-TT in DMSO andDMSO/water mixtures with increasing water fractions (fw) from 10% to90%. (B) Change in PL intensity of MTPE-TT versus water fraction inDMSO/water mixtures. Excitation at 610 nm.

FIG. 14 shows photoluminescence (PL) spectrum of MTPE-TT in the solidstate.

FIG. 15 shows mass spectrum of TPE-TPA-TT.

FIG. 16 shows ¹H-NMR spectrum of TPE-TPA-TT in CDCl₃.

FIG. 17 shows ¹³C-NMR spectrum of TPE-TPA-TT in CDCl₃.

FIG. 18 shows absorption spectrum of TPE-TPA-TT in THF.

FIG. 19 shows (A) photoluminescence (PL) spectra of TPE-TPA-TT in THFand THF/water mixtures with increasing water fractions (fw) from 10% to90%. (B) Change in PL intensity of TPE-TPA-TT versus water fraction inTHF/water mixtures. Excitation at 670 nm.

FIG. 20 shows photoluminescence (PL) spectrum of TPE-TPA-TT in the solidstate.

FIG. 21 shows mass spectrum of PTZ-BT-TPA.

FIG. 22 shows ¹H-NMR spectrum of PTZ-BT-TPA in CDCl₃.

FIG. 23 shows ¹³C-NMR spectrum of PTZ-BT-TPA in CDCl₃.

FIG. 24 shows absorption spectrum of PTZ-BT-TPA in THF.

FIG. 25 shows (A) photoluminescence (PL) spectra of PTZ-BT-TPA in THFand THF/water mixtures with increasing water fractions (fly) from 10% to90%. (B) Change in PL intensity of PTZ-BT-TPA versus water fraction inTHF/water mixtures. Excitation at 500 nm.

FIG. 26 shows photoluminescence (PL) spectrum of PTZ-BT-TPA in the solidstate.

FIG. 27 shows photographs of (a) PTZ-BT-TPA in THF and THF/watermixtures with increasing water fractions (fw) from 10% to 90%, and (b)the solid sample taken under 365 nm UV-light illumination.

FIG. 28 shows bright-field, fluorescent, and merged image of living HeLacells incubated with PTZ-BT-TPA dots for 12 h at 37° C. λ_(ex): 489 nm.

FIG. 29 shows mass spectrum of NPB-TQ.

FIG. 30 shows ¹H-NMR spectrum of NPB-TQ in CDCl₃.

FIG. 31 shows ¹³C-NMR spectrum of NPB-TQ in CDCl₃.

FIG. 32 shows absorption spectrum of NPB-TQ in THF.

FIG. 33 shows (A) photoluminescence (PL) spectra of NPB-TQ in THF andTHF/water mixtures with increasing water fractions (fw) from 10% to 90%.(B) Change in PL intensity of NPB-TQ versus water fraction in THF/watermixtures. Excitation at 610 nm.

FIG. 34 shows mass spectrum of TPE-TQ-A.

FIG. 35 shows ¹H-NMR spectrum of TPE-TQ-A in CDCl₃.

FIG. 36 shows ¹³C-NMR spectrum of TPE-TQ-A in CDCl₃.

FIG. 37 shows absorption spectrum of TPE-TQ-A in THF.

FIG. 38 shows (A) photoluminescence (PL) spectra of TPE-TQ-A in THF andTHF/water mixtures with increasing water fractions (fw) from 10% to 90%.(B) Change in PL intensity of TPE-TQ-A versus water fraction inTHF/water mixtures. Excitation at 530 nm.

FIG. 39 shows photoluminescence (PL) spectrum of TPE-TQ-A in the solidstate.

FIG. 40 shows photographs of (a) TPE-TQ-A in THF and THF/water mixtureswith increasing water fractions (fw) from 10% to 90%, and (b) the solidsample taken under 365 nm UV-light illumination.

FIG. 41 shows mass spectrum of MTPE-BTSe.

FIG. 42 shows ¹H-NMR spectrum of MTPE-BTSe in CDCl₃.

FIG. 43 shows absorption spectrum of MTPE-BTSe in THF.

FIG. 44 shows (A) photoluminescence (PL) spectra of MTPE-BTSe in THF andTHF/water mixtures with increasing water fractions (fw) from 10% to 90%.(B) Change in PL intensity of MTPE-BTSe versus water fraction inTHF/water mixtures. Excitation at 690 nm.

FIG. 45 shows photoluminescence (PL) spectrum of MTPE-BTSe in the solidstate.

FIG. 46 shows HRMS of DCDPP-2TPA.

FIG. 47 shows ¹H NMR spectrum of DCDPP-2TPA in CD₂Cl₂.

FIG. 48 shows ¹³C NMR spectrum of DCDPP-2TPA in CD₂Cl₂.

FIG. 49 shows UV-vis spectrum of DCDPP-2TPA in THF.

FIG. 50 shows (A) PL spectra of DCDPP-2TPA in THF/water mixtures withdifferent water contents, concentration is 10⁻⁵ M, λ_(ex)=408 nm; (B) PLintensities of DCDPP-2TPA in THF/water mixtures with different watercontents, insert: Photographs of the powder of DCP-2TPA taken under 365nm UV light.

FIG. 51 shows HRMS of DCDPP-2TPA4M.

FIG. 52 shows ¹H NMR spectrum of DCDPP-2TPA4M in CDCl₃.

FIG. 53 shows ¹³C NMR spectrum of DCDPP-2TPA4M in CDCl₃.

FIG. 54 shows UV-vis spectrum of DCDPP-2TPA4M in THF.

FIG. 55 shows (A) PL spectra of DCDPP-2TPA4M in THF/hexane mixtures withdifferent hexane contents, concentration is 10⁻⁵ M, λ_(ex)=439 nm; (B)PL intensities of DCDPP-2TPA4M in THF/hexane mixtures with differentwater contents, insert: Photographs of the powder of DCP-2TPA4M takenunder 365 nm UV light.

FIG. 56 shows HRMS of DCDP-2TPA.

FIG. 57 shows ¹H NMR spectrum of DCDP-2TPA in CD₂Cl₂.

FIG. 58 shows ¹³C NMR spectrum of DCDP-2TPA in CD₂Cl₂.

FIG. 59 shows UV-vis spectrum of DCDP-2TPA in THF.

FIG. 60 shows (A) PL spectra of DCDP-2TPA in THF/water mixtures withdifferent water contents, concentration is 10⁻⁵ M, =574 nm; (B) PLintensities of DCDP-2TPA in THF/water mixtures with different watercontents.

FIG. 61 shows HRMS of DCDP-2TPA4M.

FIG. 62 shows ¹H NMR spectrum of DCDP-2TPA4M in CDCl₃.

FIG. 63 shows ¹³C NMR spectrum of DCDP-2TPA4M in CDCl₃.

FIG. 64 shows UV-vis spectrum of DCDP-2TPA4M in THF.

FIG. 65 shows (A) PL spectra of DCDP-2TPA4M in THF/water mixtures withdifferent water contents, concentration is 10⁻⁵ M, =570 nm; (B) PLintensities of DCDP-2TPA4M in THF/water mixtures with different watercontents.

FIG. 66 shows (A) molecular structure of TTB, TTA and TTS. (B) PLspectra of TTS in THF/water mixtures with different water fractions(f_(w)). (C) Plots of relative PL intensity (I/I₀) versus thecomposition of THF/water mixtures of TTS. I₀=emission intensity in pureTHF solution. Concentration: 10 μM; excitation wavelength: 480 nm.Inset: fluorescent photographs of TTS in THF (f_(w)=0%) and theTHF/water mixture with f_(w)=90% taken under 365 nm UV illumination.

FIG. 67 shows normalized absorption spectra of (A) TTB, (B) TTA and (C)TTS in solvents with different polarities.

FIG. 68 shows (A) emission spectra of TTB, TTS and TTA in solvents withdifferent polarities. Concentration: 10 μM; excitation wavelength: 480nm. (B) Plots of fluorescence intensity of TTB, TTA or TTS in differentsolvents (I) versus emission intensity in toluene (I_(Tol)). (C)fluorescent photographs of TTB, TTA and TTS in different solvents takenunder 365 nm UV illumination

FIG. 69 shows (A) chemical structure of TTA. (B) UV spectrum of TTA inTHF solution. Concentration: 10 μM. (C) PL spectra of TTA in THF/watermixtures with different water fractions (f_(w)). (D) Plots of relativePL intensity (I/I₀) versus the composition of THF/water mixtures of TTA.I₀=emission intensity in pure THF solution. Concentration: 10 μM;excitation wavelength: 480 nm. Inset: fluorescent photographs of TTA inTHF=0%) and the THF/water mixture with f_(w)=90% taken under 365 nm UVillumination.

FIG. 70 shows the schematic illustration of AIE dot fabrication.

FIG. 71 shows (A) particle size distribution studied by dynamical lightscattering (DLS) and (B) absorption and emission spectra of TTS dotssuspended in water; λ_(ex)=488 nm.

FIG. 72 shows (A) photostability of TTS dots under continuous scanningat 480 nm using 450 W Xe lamp. I₀ is the initial PL intensity, while Iis that of the corresponding sample after a designated time interval.(B) Cell viability of A549 cells after incubation with 0, 0.01, 0.1, 1,and 10 μg/mL TTS dots for 24 hours, 48 hours, respectively.

FIG. 73 shows two-photon cross section of TTS in THF/water suspension(f_(w)=99% water) with a concentration of 10 μM at different excitationwavelength.

FIG. 74 shows (A) fluorescent image of the mouse after dissection. (B)Ex vivo one-photon confocal fluorescent images and two-photon confocalfluorescent images of the liver of a mouse intravenously injected TTSdots. (C) Quantitative fluorescence intensity distribution of the liverfrom the mice sacrificed at 3 hours post-injection with TTS dots atdifferent penetration depth from 0 to 300 μm.

FIG. 75 shows (A) the ex vivo bright field (up), fluorescent images(middle) and overlay images (down) of the major organs (heart, liver,spleen, lungs and kidney) of the sacrificed mice at 3 hourspost-injection with TTS dots. (B) Average fluorescence intensitydistribution for internal organs from mice sacrificed at 3 hourspost-injection with TTS dots.

FIG. 76 shows (A-E) one-photon confocal luminescence and (F-J)two-photon scanning luminescence images of the intravenously injectedTTS dots in the blood vessel of the ear of a mouse (penetration depth:100-140 μm). Scale bar: 100 μm.

FIG. 77 shows (A) one-photon fluorescent images (pseudo-color) of theblood vessel of the ear of the mouse after intravenously injection ofTTS dots (z-stage at 110 μm). (B) A zoom-in image of the selected zonein A. (C) A cross-sectional intensity profile measured along the greenline in B. (D) Two-photon fluorescent images (pseudo-color) of the bloodvessel of the ear of the mouse after intravenously injection of TTS dots(z-stage at 110 μm). (E) A zoom-in image of the selected zone in D. (F)A cross-sectional intensity profile measured along the green line in E.Scale bar: 100 μm.

FIG. 78 shows (A-C) representative two-photon fluorescent images of thebrain blood vessels of a mouse after 0.5 h injection of the TTS dotswith different penetration depths. (D-F) Representative time-lapseimages of the brain blood vessels of a mouse after 0.5 hour injection ofthe TTS dots at different monitoring time points at depth of 200 μm.Scale bar: 200 μm. (G) The reconstructed 3D image of the blood vesselsof the mouse brain after 0.5 hour injection of the TTS dots. Scale bar:150 μm.

FIG. 79 shows ¹H NMR spectrum of ROpen-DTE-TPECM in CDCl₃ at 298 K.

FIG. 80 shows HRMS of ROpen-DTE-TPECM.

FIG. 81 shows (a) PL spectra of ROpen-DTE-TPECM in THF/water mixturewith various water fractions. (b) Plot of I/I₀ versus water fraction. I₀and I are the peak PL intensities of ROpen-DTE-TPECM (10 μM) in pure THFand THF/water mixtures, respectively. Inset shows the photographs ofROpen-DTE-TPECM in THF/water mixtures with different water fractionstaken under UV illumination.

FIG. 82 shows absorption PL spectra of (a) ROpen-DTE-TPECM (THFsolution) under UV light (365 nm) irradiation and (b) RClosed-DTE-TPECM(THF solution) under red light (610 nm) irradiation for different time.(c) The absorption intensity at 650 nm of DTE-TPECM in THF during tencircles of red (610 nm)—UV (365 nm) light irradiation processes,indicating good photo-reversible ring opening and closing property ofthe molecule.

FIG. 83 shows DLS profiles and SEM images of (a) RClosed NPs and (b)ROpen NPs.

FIG. 84 shows (a) absorption and (b) PL spectra of RClosed NPs undervisible light (610 nm) irradiation for different times as indicated. (c)The absorption intensity at 650 nm of the NPs during ten circles ofvisible (610 nm)/UV light (365 nm) irradiation processes.

FIG. 85 shows (a) photoacoustic (PA) spectra of RClosed and ROpen NPs.(b) PA intensities of RClosed and ROpen NPs at 700 nm as a function ofmolar concentration based on DTE-TPECM molecules. (c) PA amplitudes ofRClosed NPs as a function of number of laser pulses (1.8×10⁴ pulses; 1.5W cm⁻² laser and 20 Hz pulse repetition rate).

FIG. 86 shows (a) PL excitation mapping and g, fluorescence decay curveof ROpen NPs. (b) Normalized PA intensities and relative fluorescencequantum yields based on the same molar extinction coefficients at 680 nmof various agents. (c) Plot of I/I₀ versus light irradiation time. Theaqueous solution of RClosed NPs or ROpen NPs (10 μM based on DTE-TPECM)was exposed to 610 nm red light and/or 365 nm UV light. I₀ and I are thePL intensity of DCF at 525 nm before and after light irradiation atdesignated time intervals.

FIG. 87 shows (a) representative PA images of subcutaneous tumor from aliving mouse after intravenous administration of RClosed NPs (800 μMbased on RClosed-DTE-TPECM, 100 μL) at designated time intervals. (b)Plot of PA intensity at 700 nm in tumor versus time post-injection ofRClosed NPs. Data are presented as mean±standard deviation (SD) (n=3mice). (c) Representative brightfield images of RClosed NPs-treatedtumor-bearing mice before and after surgery as well as representativefluorescence images of mice with complete surgical resection of tumors,followed by 610 nm red light irradiation at the operative incision sitefor 5 min. FL: fluorescence; IT: irradiation time. (d) H&E stainedtissues at the operative incision site in (c) indicate no residualtumors left behind. (e) Representative fluorescence images of RClosedNPs-treated mice with residual tumors post-surgery. The operativeincision site was irradiated by 610 nm red light for different timepoints. The red dashed circles in (c) and (e) indicate thetumor/operative incision site. The red arrow shows the residual tumorswith a diameter below 1 mm. (f) H&E stained tissues at the operativeincision site in (e) confirm the existence of residual tumors. (g)Average fluorescence intensity of residual tumors and surrounding normaltissues in (e) (n=5 mice). ** represents P<0.01, in comparison betweenresidual tumor and normal tissue. (h) Time-dependent bioluminescenceimaging of residual tumors from mice in different groups. The tumorswere debulked on day 0. The 4T1 cancer cells express luciferase,permitting bioluminescence imaging. The black arrows indicate theresidual tumor. DS: debulking surgery. (i) Quantitative analysis ofbioluminescence intensities of residual tumors from mice with varioustreatments as indicated. ** in (i) represent P<0.01, in comparisonbetween “DS+NPs+Light” cohort and other groups.

DETAILED DESCRIPTION Definitions

The following definitions are provided for the purpose of understandingthe present subject matter and for constructing the appended patentclaims.

It is noted that, as used in this specification and the appended claims,the singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

“Aggregation-induced emission”, or AIE, means thefluorescence/phosphorescence is turned on upon aggregation formation orin the solid state. When molecularly dissolved, the material isnon-emissive, but emission is turned on when intramolecular rotation isrestricted.

“Emission intensity” refers to the magnitude offluorescence/phosphorescence normally obtained from a fluorescencespectrometer or fluorescence microscopy measurement; “fluorophore” or“fluorogen” refers to a molecule which exhibits fluorescence;“luminogen” or “luminophore” refers to a molecule which exhibitsluminescence; and “AIEgen” refers to a molecule exhibiting AIEcharacteristics.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently described subject matter pertains.

Where a range of values is provided, for example, concentration ranges,percentage ranges, or ratio ranges, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the described subject matter. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and such embodiments are alsoencompassed within the described subject matter, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the described subject matter.

Throughout the application, descriptions of various embodiments use“comprising” language. However, it will be understood by one of skill inthe art, that in some specific instances, an embodiment canalternatively be described using the language “consisting essentiallyof” or “consisting of”.

For purposes of better understanding the present teachings and in no waylimiting the scope of the teachings, unless otherwise indicated, allnumbers expressing quantities, percentages or proportions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, each numerical parametershould at least be construed in light of the number of reportedsignificant digits and by applying ordinary rounding techniques.

Luminogens for Biological Applications

According to the present subject matter, certain compounds have beendeveloped exhibiting a background emission successfully suppressed inline with the expectation of several hundred fold of enhancement in theaggregated state. Following this strategy, many red or NIR emitters withAIE features can be rationally designed in accordance with the followingdescription.

In this regard, in an embodiment, the present subject matter is directedto a compound comprising a donor and an acceptor, wherein each acceptoris independently selected from the group consisting of:

wherein each D and D′ is the donor and is independently selected fromthe group consisting of:

wherein each X and X′ is independently selected from the groupconsisting of: O, S, Se, and Te;

wherein each R, R′, R″, R′″, and R″″ is independently selected from thegroup consisting of: F, H, alkyl, unsaturated alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, aminogroup, sulfonic group, alkylthio, alkoxy group, and

and wherein when any of R, R′, R″, R′″, and R″″ is a terminal functionalgroup, then each terminal functional group R, R′, R″, R′″, and R″″ isindependently selected from the group consisting of N₃, NCS, SH, NH₂,COOH, alkyne, N-Hydroxysuccinimide ester, maleimide, hydrazide, nitronegroup, —CHO, —OH, halide, and charged ionic group;

wherein each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ may be substitutedor unsubstituted and each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isindependently selected from the group consisting of H, alkyl,unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, C_(m)H_(2m+1), C₁₀H₇, C₁₂H₉, OC₆H₅, OC₁₀H₇, OC₁₂H₉,C_(m)H_(2m)COOH, C_(m)H_(2m)NCS, C_(m)H_(2m)N₃, C_(m)H_(2m)NH₂,C_(m)H_(2m)SO₃, C_(m)H_(2m)Cl, C_(m)H_(2m)Br, C_(m)H_(2m)I,

wherein n=0-2; and

wherein m=0 to 20.

For example, in an embodiment, the amino group could be used for furtherbioconjugation, such as for DNA, peptide, protein, etc.

In an embodiment of the present subject matter, at least one donor andat least one acceptor are arranged in an order selected from the groupconsisting of:

-   -   Donor-Acceptor,    -   Donor-Acceptor-Donor,    -   Acceptor-Donor-Acceptor,    -   Donor-Donor-Acceptor-Donor-Donor,    -   Acceptor-Acceptor-Donor-Acceptor-Acceptor,    -   Donor-Acceptor-Donor-Acceptor-Donor, and    -   Acceptor-Donor-Acceptor-Donor-Acceptor.

In an embodiment, the compound comprises a structure of:

wherein each X′ is independently selected from the group consisting of:S, Se, and Te;

wherein each R, R′, and R″ is independently selected from the groupconsisting of: F, H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,sulfonic group, alkylthio, and alkoxy group; and wherein when any of R,R′, and R″ is a terminal functional group, then each terminal functionalgroup R, R′, and R″ is independently selected from the group consistingof N₃, NCS, SH, NH₂, COOH, alkyne, N-Hydroxysuccinimide ester,maleimide, hydrazide, nitrone group, —CHO, —OH, halide, and chargedionic group.

In an embodiment, the compound comprises a structure of:

In an embodiment, the compound is selected from the group consisting of:

In an embodiment, the compound exhibits aggregation-induced emission(AIE).

In an embodiment, the present subject matter is directed to a probecomprising the present compound, wherein the probe is a farred/near-infrared (FR/NIR) fluorescent probe. In an embodiment, thecompound is functionalized with special targeted groups to imagebiological species. In an embodiment, the compound is fabricated in aPEG matrix and works in a form of nanoparticles. In an embodiment, thenanoparticles can be incubated with cells and used to image cellularcytoplasms.

In an embodiment, the present compound is a pyrazine-basedred/near-infrared AIEgen wherein each acceptor is independently selectedfrom the group consisting of:

wherein each D and D′ is the donor and is independently selected fromthe group consisting of:

wherein n=0-2;

wherein each X is independently O or S;

wherein each R, R′, and R″ is independently selected from the groupconsisting of: F, H, alkyl, unsaturated alkyl, heteroalkyl,heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,alkylthio, sulfonic group, and alkoxy group; and wherein when any of R,R′, and R″ is a terminal functional group, then each terminal functionalgroup R, R′, and R″ is independently selected from the group consistingof methoxyl, tertiary butyl, N₃, NCS, SH, NH₂, COOH, alkyne,N-Hydroxysuccinimide ester, maleimide, hydrazide, nitrone group, —CHO,—OH, halide, and charged ionic group.

In an embodiment, each D and D′ is

wherein n=0-2;

wherein each R, R′, and R″ is independently selected from the groupconsisting of: F, H, alkyl, unsaturated alkyl, heteroalkyl,heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,alkylthio, sulphonic group, and alkoxy group; and wherein when any ofeach R, R′, and R″ is a terminal functional group, then each terminalfunctional group R, R′, and R″ is independently selected from the groupconsisting of methoxyl, tertiary butyl, N₃, NCS, SH, NH₂, COOH, alkyne,N-Hydroxysuccinimide ester, maleimide, hydrazide, nitrone group, —CHO,—OH, halide, and charged ionic group.

In an embodiment, the compound comprises a structure of:

wherein n=1-3;

wherein each R, R′, R″, and R′″ is independently selected from the groupconsisting of: F, H, alkyl, unsaturated alkyl, heteroalkyl,heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,alkylthio, sulfonic group, and alkoxy group; and wherein when each R,R′, and R″ is a terminal functional group, then each R, R′, and R″ isindependently selected from the group consisting of methoxyl, tertiarybutyl, N₃, NCS, SH, NH₂, COOH, alkyne, N-Hydroxysuccinimide ester,maleimide, hydrazide, nitrone group, —CHO, —OH, halide, and chargedionic group.

In an embodiment, the compound is

In an embodiment, the compound exhibits aggregation-induced emission(AIE).

In an embodiment, the present subject matter is directed to a probecomprising the present compound, wherein the probe is ared/near-infrared fluorescent probe. In an embodiment, the presentcompound is functionalized with special targeted groups to imagebiological species. In an embodiment, the present compound is fabricatedin PEG/BSA and any amphiphilic molecule matrix, wherein the probe worksin a form of nanoparticles. In an embodiment, the nanoparticles areincubated with cells or tissue and used for imaging cellular cytoplasmsor tissue. In an embodiment, the nanoparticles are injected into a bloodvessel and used for blood vessel imaging.

In an embodiment, the present compound is DTE-TPECM having a structureof

In an embodiment, the structure is ROpen-DTE-TPECM and the presentcompound exhibits AIE characteristics and is used for in vivofluorescence imaging. In an embodiment, the structure isRClosed-DTE-TPECM and the present compound is used as a photoacousticagent.

In an embodiment, the present subject matter is directed to a compoundcomprising a backbone structure selected from the group consisting of:

wherein each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ may be substitutedor unsubstituted and each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isindependently selected from the group consisting of H, alkyl,unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, C_(m)H_(2m+1), C₁₀H₇, C₁₂H₉, OC₆H₅, OC₁₀H₇, OC₁₂H₉,C_(m)H_(2m)COOH, C_(m)H_(2m)NCS, C_(m)H_(2m)N₃, C_(m)H_(2m)NH₂,C_(m)H_(2m)SO₃, C_(m)H_(2m)Cl, C_(m)H_(2m)Br, C_(m)H_(2m)I,

wherein m=0 to 20; and

wherein X is independently selected from S and Se.

In an embodiment, the present compound is TTS having a structure of:

In an embodiment, the present subject matter is directed to a probecomprising the present compound, wherein the probe is used for brainvascular imaging, sentinel lymph node mapping, and tumor imaging.

Far-Red/Near-Infrared AIE Luminogens for Biological Applications

In the present subject matter, far red/near-infrared donor-acceptor(D-A) type fluorescent probes with aggregation-induced emission (AIE)features have been developed and investigated. The photophysicalproperties (absorption and photoluminescence spectra) can be simplytuned by changing the electron donating or/and withdrawing property ofthe donor or/and acceptor units. These luminophores exhibit maximalluminescence peaks from the far red to the near-infrared (FR/NIR)spectral region (650-1000 nm), and high brightness in the solid state.In the solution state, these molecules are less emissive because therotation of the aromatic rotors non-radiatively dissipates the excitonenergy. While in the aggregate state, the emission of these AIEgens isinduced or rejuvenated by the restriction of intramolecular motions(RIM) and the highly twisted molecular conformation that hampers theintermolecular π-π stacking interaction. These FR/NIR AIEgens are highlydesirable for in-vivo biological applications due to the features ofdeep-tissue penetration, less photo-damage to the body, and highsignal-to-noise ratio of FR/NIR light. Primary experiments indicate thatthese FR/NIR dyes are excellent candidates for biology-relatedapplications.

For example, dyes MTPE-TP and PTZ-BT-TPA have been tested in living HeLacells in order to analyze their use as biosensors for bioimaging. Thedye and an amphiphilic polymer were used to fabricate fluorescent dots,and the dots can accumulate preferentially in tumors through theenhanced permeability and retention (EPR) effect. As shown in FIGS. 8and 28, an intense red fluorescence was observed in the cellularcytoplasms, demonstrating the great potential of AIE dots as an FR/NIRfluorescent probe for biological imaging. Some strong donor-acceptorsystems (e.g., PTZ-BT-TPA, TPE-TPA-TT, and NPB-TQ) exhibit excellenttwo-photon absorption properties, which demonstrate their applicationfor two-photon and multiphoton fluorescence imaging.

In addition, by the introduction of special functional and targetedgroups in the AIEgens, the dyes can be used for specific targeting ofbiological species. For example, there are two alkyne groups in the AIEdye TPE-TQ-A, which is convenient for further introduction of functionalor targeted groups through click reaction.

Characterization

¹H and ¹³C NMR spectra were measured on a Bruker ARX 400 NMRspectrometer using chloroforms solvent and tetramethylsilane (TMS, δ=0)as internal reference. High-resolution mass spectra (HRMS) were recordedon a Finnigan MAT TSQ 7000 Mass Spectrometer System operated in aMALDI-TOF mode. Absorption spectra were recorded on a Shimadzu UV-3600spectrophotometer. Steady-state fluorescence spectra were recorded on aPerkin Elmer LS 55 spectrometer or a HORIBA Spectrofluorometer. Laserconfocal scanning microscope images were collected on Zeiss laserscanning confocal microscope (LSM7 DUO) and analyzed using ZEN 2009software (Carl Zeiss).

Fabrication of AIE Dots

Generally, 1 mg of the AIEgen and 5 mg of DSPE-PEG-Biotin were dissolvedin 1 mL of chloroform. Subsequently, 9 mL of Milli-Q water was addedinto the chloroform solution under sonication for 10 minutes at roomtemperature using a microtip probe sonicator with an 18 W output(XL2000, Misonix Incorp., USA). The mixture was stirred in the nitrogenflow overnight until the chloroform was completely evaporated. A clearsolution was obtained, and the solution was filtered through a 0.45 μmmicrofilter to result in the formation of AIE dots.

Cell Culture and Staining

Cell Culture: HeLa cells were cultured in MEM containing 10% FBS andantibiotics (100 units/mL penicillin and 100 g/mL streptomycin) in a 5%CO₂ humidity incubator at 37° C. All culture media were supplementedwith 10% heat-inactivated FBS, 100 units/mL penicillin, and 100 μg/mLstreptomycin. Before the experiment, the cells were precultured untilconfluence was reached.

Cell Imaging: HeLa cells were grown overnight on a 35 mm petri dish witha cover slip at 37° C. After removal of the medium, the adherent cellswere washed twice with 1× phosphate buffered saline (PBS) buffer. TheAIE dots were then added to the chamber. After incubation for 12 hours,the cells were washed three times with 1×PBS buffer. The cells wereimaged on a Zeiss laser scanning confocal microscope and analyzed usingZEN 2009 software.

AIE Study

The dyes MTPE-TP and MTPE-TT showed typical twisted intramolecularcharge transfer (TICT) and AIE features when the water fractions wereincreased in the DMSO/water mixtures (FIGS. 5 and 13). The dyesTPE-TPA-TT, PTZ-BT-TPA, NPB-TQ, TPE-TQ-A, and MTPE-BTSe showed typicaltwisted intramolecular charge transfer (TICT) and AIE features when thewater fractions were increased in the THF/water mixtures as shown in(FIGS. 19, 25, 33, 38, and 44). Initially, a small amount of water wasadded, and the fluorescence decreased, a typical TICT effect arisingfrom increased solvent polarity. When more water was added, the AIEmolecules formed nanoaggregates, and the emission increased. Thesolutions of the AIEgens were slightly emissive in solution, whilestrongly emissive in the aggregation state of 90% water fraction.

Pyrazine-Containing Red/Near-Infrared AIE Luminogens

In the present subject matter, pyrazine-containing red and near-infraredAIEgens having D-A structures are generated in simple synthetic steps.Pyrazine or dipyrazine are used as acceptors to tune the electronaccepting property, while triphenylamine, carbazole, and theirderivatives or other groups are utilized as donors to tune the electrondonating property, which enables the photo-physical property of AIEgens.The resulting AIEgens are conjugated with whole aromatic structureswherein no double bond is involved, thus making the luminogens much morestable than common, conventional luminogens.

The AIEgens show maximum emissions ranging from 630-900 nm, covering thespectral range from red to near-infrared light. Due to featuresincluding deep tissue penetration, negligible biologicalauto-fluorescence, low photo-damage, and high signal-to noise ratio,etc., the AIEgens exhibit huge application potential in preclinicalresearch and clinical practice for noninvasive real-time tumor diagnosisand image-guided cancer therapy. Although no molecular rotator of TPE isincorporated into the structure, all of the luminogens are stillAIE-active because they possess twisted and flexible molecularconformation.

In solution state, the luminogens are non-emissive or less emissive,because intramolecular motion easily takes place to dissipate theexcitation state energy non-radiatively, whereas in the aggregatedstate, such motion is suppressed to open up the radiative transitionchannel. Meanwhile, the twisted conformation of pyrazine-containedluminogens may prohibit the formation of π-π stacking when molecules areclosely located. Both factors collectively determine a remarkablyenhanced emission in the aggregate state. The AIE behavior ofpyrazine-contained luminogens may facilitate the fabrication of highlyemissive nanoparticles with excellent stability, which is extremelydesirable for biological applications.

Characterization

¹H and ¹³C NMR spectra were measured with a Bruker AVIII 400spectrometer using CDCl₃ or CD₃Cl₂ as the solvent. When CDCl₃ was used,the tetramethylsilane (TMS; δ=0) was used as the internal reference.High resolution mass spectra (HRMS) were recorded using a GCT premierCAB048 mass spectrometer operated in MALDI-TOF mode. Absorption spectrawere recorded on a Varian Cary 50 spectrophotometer. Steady-statefluorescence spectra were measured using a Perkin Elmer LS 55spectrometer or a HORIBA Spectrofluorometer.

Some of the developed luminogens exhibited typical AIE properties,whereas others displayed weak twisted intramolecular charge transfer(TICT) effects plus strong AIE effects. For example, DCDPP-2TPA4M givesno emission when molecules are dissolved in THE After addition of thepoor solvent of hexane, no emission is observed when the hexane fractionreaches 60%. However, a remarkable enhanced emission can be detectedwhen further increasing the hexane fraction. Because hexane is a poorsolvent, the addition of a large amount of hexane into the THF solutionmust induce the formation of aggregates, thus giving rise to the AIEphenomenon (FIG. 55). The AIE effect can be also detected in aggregatesof DCDP-2TPA formed in high water fractions of THF/water mixtures, and avery weak emission can be found when molecules are dispersed in THF,which is due to the TICT effect (FIG. 60).

Design and Synthesis of Red AIEgens for In Vivo Deep-Tissue Imaging

The present subject matter relates to a design strategy for ultra brightred luminogens with aggregation-induced emission (AIE) features in astrong donor-acceptor (D-A) system and use of said strategy for thedevelopment of AIE luminogens for in vivo deep-tissue imaging bytwo-photon imaging technique with high performances. In particular, thepresently described subject matter relates to luminogens comprisingtetraphenylethene derivatives featured with aggregation-induced emissioncharacteristics (AIE).

In one embodiment, these luminogens have arylamine as the electron donorand benzothiadiazole or benzoselenadiazole as the electron acceptor.Moreover, the strategy sheds light on the development of efficientsolid-state red/NIR emitters with high brightness, large Stokes shift,good biocompatibility, satisfactory photostability and high two-photonabsorption cross section for high contrast in vivo deep-tissue imaging.

In one embodiment, the present subject matter generally relates to adesign strategy for ultra bright red luminogens with aggregation-inducedemission (AIE) features based on the presence of a TPE group, anarylamine, and a benzothiadiazole or benzoselenadiazole unit. Due toenhanced charge transfer (CT) effect, the background emission in THF ofred luminogen is successfully suppressed using such compounds, withseveral hundred fold of emission enhancements in the aggregated state.Moreover, thanks to the highly emissive feature of TPE derivatives inthe solid state, in vivo deep-tissue imaging, including brain vascularimaging, sentinel lymph node mapping and tumor imaging, with highcontrast and high penetration depth are achieved.

In an embodiment, the present subject matter relates to use of an AIEmaterial as a fluorescent probe for brain vascular imaging, sentinellymph node mapping and tumor imaging.

In another embodiment, the present subject matter relates to use of anAIE material as fluorescent probe with good biocompatibility.

The photoluminescence (PL) spectrum of TTS (FIG. 66) is basically a flatline parallel to the abscissa in pure THF. The spectral pattern remainsunchanged at water content up to 40%. Afterwards, the PL intensityincreases dramatically. The higher the water fraction, the stronger theemission intensity. The emission peak reaches to ˜636 nm. The emissionintensity reaches its maximum value at 90% water content, which is170-fold higher than that in pure THF

Solution

FIG. 67 shows the UV spectrum of TTB, TTA, and TTS measured in differentsolvents. With introduction of extra arylamine units to the TTBmolecule, the absorption peak of TTS is slightly red-shifted due tolonger electronic conjugation. The absorption peaks of TTB, TTA, and TTSare not significantly affected by the solvent polarity.

TABLE 1 Absorption and emission of TTB, TTA and TTS in differentsolvents. TTB TTA TTS λ_(ab) λ_(em) λ_(ab) λ_(em) λ_(ab) λ_(em) Solvent(nm)^([a]) (nm)^([b]) (nm)^([a]) (nm)^([b]) (nm)^([a]) (nm)^([b]) Tol470 596 478 620 484 623 DO 465 609 471 632 478 640 THF 469 618 474nd^([c]) 481 nd^([c]) NMP 469 654 478 nd^([c]) 484 nd^([c]) ^([a])λ_(ab)= absorption maximum. ^([b])λ_(em) = emission maximum. ^([c])nd = notdetected.

The PL spectra of TTB, TTA, and TTS measured in different solvents areshown in FIG. 68. When the solvent polarity was increased from Tol toNMP, the emission of TTB, TTA, and TTS are red-shifted with an emissionquenching effect. This suggests that these molecules have anintramolecular charge transfer (CT) effect from the electron-donatingarylamine unit to the electron-accepting benzothiadiazole. However,compared with TTB molecule, TTA and TTS molecules are more sensitive tosolvent polarity. TTA and TTS are emissive in Tol, weak emissive in DOand nonemissive in THF with medium polarity. In contrast, TTB is highlyemissive in Tol, DO, and THF, and even in the high polarity solvent NMP,TTB is still emissive observed by the naked eye. The result shows thatthe stronger D-A system with two more arylamine units fusioned to theTTB molecular structure greatly enhances the CT effect. It is probablethat even in THF with medium polarity, the conformation of the TTA orTTS becomes twisted and the charge becomes separated. Its light emissionis red-shifted in color, but significantly decreased in intensity due tothe locally excited (LE) state to twisted intramolecular charge transfer(TICT) state transition.

In an embodiment, the PL spectrum of TTA shows a similar behavior to TTS(FIG. 69). In pure THF solution, it is non-emissive. The PL intensityremains unchanged at water fraction f_(w)<50%. Afterwards, the PLintensity reaches its maximum value at 90% water content with emissionpeak at ˜635 nm. Thus, both TTS and TTA are AIE-active.

TABLE 2 Optical properties of TTS. λ_(ab) powder τ E_(g) HOMO/(nm)^([a]) soln aggr (Φ_(F))^([c]) (ns)^([d]) (eV)^([e]) LUMO(eV)^([f])TTS 481 641 636 636 1.86 2.21 −5.05 (−4.52)/ (0.1) (34.1) (2.29) −2.84(−2.23) ^([a])λ_(ab) = absorption maximum in THF. ^([b])λ_(em) =emission maximum in THF solution (soln), THF/water mixture (1:9 byvolume) (aggr). ^([c])solid powders fluorescence quantum yield (Φ_(F),%) given in the parentheses. ^([d])fluorescence lifetime of solidpowders (τ). ^([e])E_(g) = energy band gap calculated from the onset ofthe absorption spectrum. ^([f])HOMO = highest occupied molecularorbitals calculated from the onset oxidation potential, LUMO = lowestunoccupied molecular orbitals estimated by using the equation: LUMO =HOMO + Eg, the values in the parentheses are derived from theoreticalDFT calculations.

Due to the higher Φ_(F) and much better processability of TTS, it waschosen for further biological applications. For easy dispersion of thehighly hydrophobic TTS molecules in an aqueous environment, the TTS dotswere formulated through a simple procedure by a nanoprecipitation methodwith amphiphilic and biocompatible1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethyleneglycol)-2000] (DSPE-PEG 2000) as the encapsulationmatrix (FIG. 70). The size of TTS dots was measured by laser lightscattering (LLS), which indicated that the hydrodynamic diameter was˜120 nm (FIG. 71A). TTS dots have an absorption maxima at 497 nm inaqueous media, which fits well with the commercial laser excitation at488 nm. The strong emission of TTS dots (FIG. 71B) peaks at the redregion (630 nm) and tails to the near-infrared region (820 nm). It isnoteworthy that the Stokes shift of the TTS dots is big enough (>130 nm)to solve the serious problem of the self-quenching effect common inconventional organic dye molecules.

FIG. 72A shows the different fluorescence stability of AIE dots andrubrene dots at continuous irradiation with a 480 nmXe lamp. After 12minutes of irradiation, the AIE dots maintained ˜80% of their initialfluorescence intensity, which is much higher than that of rubrene dots(˜25%). The cytotoxicity of the AIE dots was evaluated by metabolicviability of A549 cells after incubation at different concentrations andtime intervals (FIG. 72B). Cell viabilities remained above 90% afterbeing treated with 0.01, 0.1, and 1 μg/mL AIE dots within the testedperiods of time. After being treated with a higher concentration (10μg/mL) AIE dots for 24 or 48 hours, ˜90% was still maintained,indicative of low cytotoxicity of the AIE dots.

The TPA spectrum of AIE dots in water with the wavelength range from800-1000 nm at 20 nm intervals was investigated. The TTS molecule inaggregate shows efficient two-photon absorption properties, giving ahigh maximum's value of 310 GM and a high quantum efficiency of 38.5%relative to rhodamine B (Φ_(F)=41.9% in methanol) under excitation of a900 nm laser light (FIG. 73). Moreover, a 900 nm fs laser expects tohave deeper penetration and better focusing capability than the commonlyused 770-860 nm Ti:Sapphire fs laser. Therefore, the TTS dots are apromising candidate for two-photon deep-tissue in vivo bioimaging.

To study the biodistribution of AIE-dots in mice, AIE-dots wereintravenously injected from a tail vein. Major organs of the mouseincluding heart, liver, spleen, lungs, and kidney were resected at 3hours post-injection and imaged immediately (FIG. 74A). Due to themetabolic functions of the mouse, the average fluorescence intensity ofthe AIE-dots mainly appears in the liver (FIG. 74B). To obtain furtherproof, the excised liver was imaged using one-photon excitation (FIG.75A) and two-photon excitation. Using one-photon microscopy, the strongfluorescence from AIE dots was observed in the liver at a penetrationdepth of ˜40 μm. However, it quickly faded out at a depth of ˜80 μm anddisappeared at a penetration depth of ˜120 μm because of the significantabsorption/scattering loss of the 488 nm excitation in the liver (FIGS.75B-C). In contrast, the deeper penetration (>200 μm) and milder decayrate of fluorescence was achieved with two-photon excitation (FIGS.75B-C). Therefore, the wo-photon fluorescence imaging technique is moreadvantageous for deep-tissue imaging with higher penetration depth.

One-photon and two-photon blood vasculature imaging in a live mouse earwas investigated using AIE dots (FIG. 76). The red fluorescence wasobserved from the small capillaries at a depth of 100-140 μm. However,with the increase of depth, the two-photon signal shows an obviousadvantage over the one-photon signal with a much milder signal decayrate. For further comparison of signal difference between the one-photonand two-photon techniques, images at 110 μm were selected withpseudo-color. For one photon excitation, the signal contrast was low,indicated by the ambiguous signal from small capillaries and surroundingtissue (FIGS. 77A-C). In comparison, the image contrast wassubstantially improved using the two-photon technique. After carefulmeasurement (FIGS. 77D-F), the quantitative measurement of the diameterof the indicated tiny capillary was ˜4 μm in the deep location (110 μm).This is believed to be the first report using AIE dots for the accuratemeasurement of capillary diameter of a mouse ear. Therefore, compared tothe one-photon imaging technique, the two-photon imaging techniqueenables observation of finer details of the tiny capillaries with a highsignal-to-noise ratio.

Because TTS dots enable deep penetration and high contrast imaging bythe two-photon technique, TTS dots were further exploited in real-timeimaging of blood vessels at a deeper level of a mouse brain. FIGS. 78A-Cshow representative images of the mouse brain vasculature imaged througha cranial window at a penetration depth from 125-225 μm. Thefluorescence signal from the AIE-dots was still detectable up to 350 μm.FIGS. 78D-F are representative high contrast images of the mouse brainvasculature at different designed time intervals. The dynamic real-timeblood flow process in major blood vessels, even in other smallercapillaries, could be accurately and vividly displayed by intense redfluorescence from AIE dots. A high resolution 3D in vivo image wasreconstructed (FIG. 78G), which provides a general and clear spatialpicture about major blood vasculature networks, as well as the detailsof tiny capillaries. Thus, AIE dots have successfully realized real-timemonitoring of the dynamic blood flow process in vivo with high spatialand temporal resolution.

EXAMPLES Synthesis of MTPE-TP

The synthesis of MTPE-TP is shown below in Scheme 1.

4,4′-dimethoxybenzophenone (20 mmol, 4.84 g), 4-bromobenzophenone (24mmol, 6.26 g) and zinc dust (80 mmol, 5.2 g) were added to a 500 mLround-bottom flask. Then the flask was evacuated under vacuum andflushed with dry nitrogen three times. After addition of 250 mL ofanhydrous THF, the mixture was cooled to −10° C. and TiCl₄ (7.7 mL, 70mmol) was added dropwise and stirred for 30 minutes. The mixture washeated to reflux and stirred for 12 hours. After cooling to roomtemperature, the reaction was quenched by 1 M HCl solution, and themixture was extracted with dichloromethane three times. The organicphase was combined, dried with MgSO₄, and the solvent was evaporatedunder reduced pressure. The crude product was purified on a silica gelcolumn chromatography using dichloromethane/hexane (v/v 1:5) as theeluent to give4,4′-(2-(4-bromophenyl)-2-phenylethene-1,1-diyl)bis(methoxybenzene) as awhite solid (46% yield).

4,4′-(2-(4-Bromophenyl)-2-phenylethene-1,1-diyl)bis(methoxybenzene)(3.77 g, 8 mmol),[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II)(Pd(dppf)Cl₂) (0.22 g, 0.3 mmol), KOAc (3.0 g, 30 mmol), andbis(pinacolato)diboron (3.05 g, 12 mmol) were added into a 100 mLtwo-necked round-bottom flask, and the flask was evacuated under vacuumand flushed with dry nitrogen for three times. Anhydrous 1,4-dioxane (40mL) was added, and the mixture was heated to reflux and stirred undernitrogen atmosphere for 24 hours. Afterwards, water was added, and themixture was washed with dichloromethane three times. The organic phasewas combined, dried with MgSO₄, and the solvent was evaporated underreduced pressure. The crude product was purified by silica gel columnchromatography using dichloromethane/hexane (v/v 1:3) as the eluent toafford2-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneas a white solid (85% yield).

2-(4-(2,2-Bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(2.59 g, 5 mmol), 2,5-dibromo-3,4-dinitrothiophene (0.67 g, 2 mmol),tetrakis(triphenylphosphine)palladium (0) (Pd(PPh₃)₄) (60 mg, 0.05mmol), and K₂CO₃ (2.07 g, 15 mmol) were added into a 100 mL two-neckedround-bottom flask. The flask was vacuumed and purged with dry nitrogenthree times. Then anhydrous tetrahydrofuran (40 mL) and water (10 mL)were added, and the mixture was heated to reflux and stirred undernitrogen atmosphere for 24 hours in the absence of light. Afterwards,water was added, and the mixture was extracted with dichloromethane. Theorganic phase was combined and dried with MgSO₄. After the removal ofthe solvent under reduced pressure, the residue was purified by silicagel column chromatography using dichloromethane/hexane (v/v 1:3) as theeluent to result in2,5-bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-3,4-dinitrothiopheneas an orange solid (81% yield).

2,5-Bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-3,4-dinitrothiophene(1.43 g, 1.5 mmol) was suspended in a solution of 30 mL of ethanol and20 mL of concentrated HCl under vigorous stirring, and SnCl₂.2H₂O (6.75g, 30 mmol) was added into the mixture in three portions. Then themixture was heated to reflux and stirred under nitrogen atmosphere for20 hours. After cooling to room temperature, 100 mL of water was added.The suspension was filtered through to give2,5-bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)thiophene-3,4-diamineas a yellow-organic solid, and it was used without further purification.

2,5-Bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)thiophene-3,4-diamine(1.07 g, 1.2 mmol) was dissolved in chloroform (20 mL) and acetic acid(20 mL), and benzil (0.32 g, 1.5 mmol) was added. The mixture was heatedto reflux and stirred for 18 hours. After cooling to room temperature,water was added to the solution, and the mixture was extracted withdichloromethane three times. The organic phase was washed with brineseveral times, and dried with MgSO₄. After the removal of the solventunder reduced pressure, the residue was purified by silica gel columnchromatography using dichloromethane/hexane (v/v 1:3) as the eluent toresult in5,7-bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-2,3-diphenylthieno[3,4-b′]pyrazine(MTPE-TP) as a dark purple solid (81% yield).

¹H NMR (400 MHz, CDCl₃, δ, ppm): 8.07 (d, 4H), 7.50 (d, 4H), 7.37-7.27(m, 6H), 7.17-7.05 (m, 14H), 7.02 (d, 4H), 6.95 (d, 4H), 6.66 (dd, 8H),3.74 (d, 12H). ¹³C NMR (100 MHz, CDCl₃, δ, ppm): 158.29, 158.12, 152.17,144.23, 143.91, 140.57, 139.30, 138.79, 136.42, 136.31, 132.70, 132.68,131.95, 131.61, 131.19, 131.05, 129.87, 128.84, 128.07, 127.77, 126.81,126.20, 113.22, 113.00, 55.11. HRMS (MALDI) m/z calcd for C₇₄H₅₆O₄N₂S1068.3961, found 1068.3969.

Synthesis of MTPE-TT

The synthesis of MTPE-TT is shown below in Scheme 2.

2,5-Bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)thiophene-3,4-diamine(0.9 g, 1 mmol) was dissolved in dry pyridine (35 mL) under nitrogenatmosphere. Then N-thionylaniline (0.225 mL, 2 mmol) and Me₃SiCl (0.23mL, 1.8 mmol) were added into this solution. The reaction continued at80° C. for 18 hours. Most of the pyridine was evaporated and the residuewas purified by silica gel column chromatography on silica gel usingdichloromethane/hexane (v/v 1:3) as the eluent to give5,7-bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-2,3-diphenylthieno[3,4-b][1,2,5]thiadiazole (MTPE-TT) as a dark blue solid (76% yield).

¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.85 (d, 4H), 7.16-7.04 (m, 14H), 7.01(d, 4H), 6.94 (d, 4H), 6.65 (dd, 8H), 3.74 (d, 12H). ¹³C NMR (100 MHz,CDCl₃, δ, ppm): 158.31, 158.16, 157.49, 144.08, 143.73, 140.72, 138.63,136.30, 136.28, 132.65, 132.21, 131.52, 130.49, 127.80, 126.26, 124.96,118.77, 113.25, 113.01, 55.10. HRMS (MALDI) m/z calcd for C₆₀H₄₆O₄N₂S₂922.2857, found 922.2899.

Synthesis of TPE-TPA-TT

The synthesis of TPE-TPA-TT is shown below in Scheme 3.

4-(Diphenylamino)phenyl)boronic acid (2.89 g, 10 mmol),2,5-dibromo-3,4-dinitrothiophene (1.33 g, 4 mmol), Pd(PPh₃)₄ (93 mg,0.08 mmol), and K₂CO₃ (4.14 g, 30 mmol) were added into a 250 mLtwo-necked round-bottom flask. The flask was vacuumed and purged withdry nitrogen three times. Then anhydrous tetrahydrofuran (80 mL) andwater (20 mL) were added, and the mixture was heated to reflux andstirred under nitrogen atmosphere for 24 hours in the absence of light.After cooling to room temperature, water was added, and the mixture wasextracted with dichloromethane. The organic phase was combined, anddried with MgSO₄. After the removal of the solvent under reducedpressure, the residue was purified by silica gel column chromatographyusing dichloromethane/hexane (v/v 1:4) as the eluent to result in4,4′-(3,4-dinitrothiophene-2,5-diyl)bis(N,N-diphenylaniline) as a redsolid (81% yield).

4,4′-(3,4-Dinitrothiophene-2,5-diyl)bis(N,N-diphenylaniline) (1.98 g, 3mmol) and anhydrous tetrahydrofuran (80 mL) were added into a 250 mLtwo-necked round-bottom flask. N-Bromosuccinimide (NBS, 2.97 g, 15 mmol)was added into the solution in three portions, and the mixture wasstirred at room temperature in the absent of light. Thin layerchromatography (TLC) was used to monitor the reaction process. When thereaction was completed, aqueous NaHCO₃ solution was added into themixture. The solution was extracted with dichloromethane three times.The organic phase was combined and dried with MgSO₄. After removal ofthe solvent under reduced pressure, the residue was purified by silicagel column chromatography using dichloromethane/hexane (v/v 1:4) as theeluent to result in4,4′-(3,4-dinitrothiophene-2,5-diyl)bis(N,N-bis(4-bromophenyl)aniline)as a red solid (92% yield).

4,4′-(3,4-Dinitrothiophene-2,5-diyl)bis(N,N-bis(4-bromophenyl)aniline)(2.44 g, 2.5 mmol),4,4,5,5-tetramethyl-2-(4-(1,2,2-triphenylvinyl)phenyl)-1,3,2-dioxaborolane(5.50 g, 12 mmol), Pd(PPh₃)₄ (230 mg, 0.2 mmol), and K₂CO₃ (4.14 g, 30mmol) were added into a 250 mL two-necked round-bottom flask. The flaskwas vacuumed and purged with dry nitrogen three times. Then anhydroustetrahydrofuran (60 mL) and water (15 mL) were added, and the mixturewas heated to reflux and stirred under nitrogen atmosphere for 24 hoursin the absence of light. After cooling to room temperature, water wasadded, and the mixture was extracted with dichloromethane three times.The organic phase was combined, and dried with MgSO₄. After the removalof the solvent under reduced pressure, the residue was purified bysilica gel column chromatography using dichloromethane/hexane (v/v 1:4)as the eluent to result inN,N′-((3,4-dinitrothiophene-2,5-diyl)bis(4,1-phenylene))bis(4′-(1,2,2-triphenylvinyl)-N-(4′-(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)-[1,1′-biphenyl]-4-amine)as a red solid (78% yield).

N,N′-((3,4-dinitrothiophene-2,5-diyl)bis(4,1-phenylene))bis(4′-(1,2,2-triphenylvinyl)-N-(4′-(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)-[1,1′-biphenyl]-4-amine)(2.97 g, 1.5 mmol) was suspended in a solution of 60 mL of ethanol and40 mL of concentrated HCl under vigorous stirring in a 250 mL two-neckedround-bottom flask, and SnCl₂.2H₂O (6.75 g, 30 mmol) was added in threeportions. Then the mixture was heated to reflux and stirred undernitrogen atmosphere for 20 hours. After cooling to room temperature, 150mL of water was added. The suspension was filtered through to give2,5-bis(4-(bis(4′-(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)amino)phenyl)thiophene-3,4-diamineas a yellow-organic solid, and it was used without further purification.

2,5-Bis(4-(bis(4′-(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)amino)phenyl)thiophene-3,4-diamine(1.92 g, 1 mmol) was dissolved in dry pyridine (35 mL) under nitrogen.N-thionylaniline (0.225 mL, 2 mmol) and Me₃SiCl (0.23 mL, 1.8 mmol) wereadded to this solution. The reaction continued at 80° C. for 18 hours.Most of the pyridine was evaporated and the residue was purified bysilica gel column chromatography using dichloromethane/hexane (v/v 1:4)as the eluent to giveN,N′-(thieno[3,4-b][1,2,5]thiadiazole-5,7-diylbis(4,1-phenylene))bis(4′-(1,2,2-triphenylvinyl)-N-(4′-(1,2,2-triphenylvinyl)-[1,1′-biphenyl]-4-yl)-[1,1′-biphenyl]-4-amine)(TPE-TPA-TT) as a blue solid (71% yield).

¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.98 (d, 4H), 7.48 (d, 8H), 7.34 (d,8H), 7.17 (d, 12H), 7.14-7.01 (m, 68H). ¹³C NMR (100 MHz, CDCl₃, δ,ppm): 157.21, 146.52, 146.25, 143.80, 143.78, 143.75, 142.53, 141.08,140.57, 138.09, 135.51, 131.85, 131.44, 131.38, 127.78, 127.70, 127.66,127.35, 126.66, 126.50, 126.44, 125.75, 124.74, 123.92, 117.71. HRMS(MALDI) m/z calcd for C₁₄₄H₁₁₀N₄S₂ 1949.7423, found 1949.7253.

Synthesis of PTZ-BT-TPA

The synthesis of PTZ-BT-TPA is shown below in Scheme 4.

10H-phenothiazine (10 g, 50 mmol), 1-bromooctane (11.6 g, 60 mmol),sodium hydroxide (12 g, 300 mmol), and dimethyl sulfoxide (DMSO) (100mL) were added into a 250 mL two-necked round-bottom flask, and themixture was stirred at room temperature for 2 days. Then brine wasadded, and the mixture was extracted with ethyl acetate three times. Theorganic phase was combined and dried with MgSO₄. After removal of thesolvent under reduced pressure, the residue was purified by silica gelcolumn chromatography using dichloromethane/hexane (v/v 1:4) as theeluent to result in 10-octyl-10H-phenothiazine as a colorless oil (86%yield).

NBS (7.83 g, 44 mmol) was added to a solution of10-octyl-10H-phenothiazine (6.23 g, 20 mmol) in toluene (20 mL) andacetic acid (70 mL). The reaction mixture was stirred for 3 hours atroom temperature in the absent of light. Water was added to stop thereaction, and the mixture was extracted with ethyl acetate three times.The organic phase was combined and dried with MgSO₄. After removal ofthe solvent under reduced pressure, the residue was purified by silicagel column chromatography using dichloromethane/hexane (v/v 1:3) as theeluent to result in 3,7-dibromo-10-octyl-10H-phenothiazine as a viscousoil (91% yield).

3,7-Dibromo-10-octyl-10H-phenothiazine (2.35 g, 5 mmol),bis(pinacolato)diboron (3.18 g, 12.5 mmol), PdCl₂(dppf) (163 mg, 0.2mmol), and KOAc (1.96 g, 20 mmol) were added into a 100 mL two-neckedround-bottom flask. The flask was vacuumed and purged with dry nitrogenthree times. Then anhydrous 1,4-dioxane (40 mL) was added, and themixture was heated to 90° C. and stirred for 24 hours in the absence oflight. Afterwards, water was added, and the mixture was extracted withdichloromethane. The organic phase was combined and dried with MgSO₄.After removal of the solvent under reduced pressure, the residue waspurified by silica gel column chromatography usingdichloromethane/hexane (v/v 1:2) as the eluent to result in10-octyl-3,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-10H-phenothiazineas a colorless solid (91% yield).

4-(Diphenylamino)phenyl)boronic acid (2.89 g, 10 mmol),4,7-dibromobenzo[c][1,2,5]thiadiazole (3.53 g, 12 mmol), Pd(PPh₃)₄ (120mg, 0.1 mmol), and K₂CO₃ (2.76 g, 20 mmol) were added into a 250 mLtwo-necked round-bottom flask. The flask was vacuumed and purged withdry nitrogen three times. Then anhydrous tetrahydrofuran (80 mL) andwater (20 mL) were added, and the mixture was heated to reflux andstirred for 24 hours in the absence of light. After cooling to roomtemperature, water was added, and the mixture was extracted withdichloromethane. The organic phase was combined and dried with MgSO₄.After removal of the solvent under reduced pressure, the residue waspurified by silica gel column chromatography usingdichloromethane/hexane (v/v 1:4) as the eluent to result in4-(7-bromobenzo[c][1,2,5]thiadiazol-4-yl)-N,N-diphenylaniline as a redsolid (72% yield).

4-(7-Bromobenzo[c][1,2,5]thiadiazol-4-yl)-N,N-diphenylaniline (2.29 g, 5mmol),10-octyl-3,7-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-10H-phenothiazine(1.13 g, 2 mmol), Pd(PPh₃)₄ (60 mg, 0.05 mmol), and K₂CO₃ (2.76 g, 20mmol) were added into a 100 mL two-necked round-bottom flask. The flaskwas vacuumed and purged with dry nitrogen three times. Then anhydroustetrahydrofuran (40 mL) and water (10 mL) were added, and the mixturewas heated to reflux and stirred for 24 hours in the absence of light.After cooling to room temperature, water was added, and the mixture wasextracted with dichloromethane. The organic phase was combined and driedwith MgSO₄. After removal of the solvent under reduced pressure, theresidue was purified by silica gel column chromatography usingdichloromethane/hexane (v/v 1:3) as the eluent to result in4,4′-((10-octyl-10H-phenothiazine-3,7-diyl)bis(benzo[c][1,2,5]thiadiazole-7,4-diyl))bis(N,N-diphenylaniline)(PTZ-BT-TPA) as a red solid (78% yield).

¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.92-7.82 (m, 6H), 7.79 (d, 2H), 7.73(s, 4H), 7.34-7.26 (m, 8H), 7.24-7.15 (m, 12H), 7.10-7.01 (m, 6H), 3.98(t, 2H), 2.00-1.87 (m, 2H), 1.54-1.45 (m, 2H), 1.41-1.23 (m, 8H), 0.88(t, 3H). ¹³C NMR (100 MHz, CDCl₃, δ, ppm): 154.11, 154.04, 148.02,147.49, 144.80, 132.41, 131.79, 131.32, 130.94, 129.92, 129.38, 128.31,127.80, 127.37, 127.22, 124.92, 124.39, 123.32, 122.91, 115.20, 47.80,31.80, 31.61, 29.30, 27.09, 26.92, 22.67, 14.15. HRMS (MALDI) m/z calcdfor C₆₈H₅₅N₇S₃ 1065.3681, found 1065.3640.

Synthesis of NPB-TQ

The synthesis of NPB-TQ is shown below in Scheme 5.

N-phenylnaphthalen-1-amine (5.28 g, 24 mmol), 1-bromo-4-iodobenzene(5.66 g, 20 mmol), sodium tert-butoxide (2.3 g, 24 mmol), and palladium(II) acetate (Pd(OAc)₂) (225 mg, 1 mmol) were added into a 250 mLtwo-necked round-bottom flask. The flask was vacuumed and purged withdry nitrogen three times. Then anhydrous toluene (60 mL) andtri-tert-butylphosphine (Bu₃P, 1.5 mmol, 1 M toluene solution, 1.5 mL)were added, and the mixture was heated to reflux and stirred for 24hours in the absence of light. After cooling to room temperature, waterwas added, and the mixture was extracted with dichloromethane. Theorganic phase was combined and dried with MgSO₄. After removal of thesolvent under reduced pressure, the residue was purified by columnchromatography on silica gel using dichloromethane/hexane (v/v 1:5) asthe eluent to result in N-(4-bromophenyl)-N-phenylnaphthalen-1-amine asa colorless solid (76% yield).

N-(4-Bromophenyl)-N-phenylnaphthalen-1-amine (5.61 g, 15 mmol),[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II)(Pd(dppf)Cl₂) (0.36 g, 0.5 mmol), KOAc (5.0 g, 50 mmol), andbis(pinacolato)diboron (6.1 g, 24 mmol) were added into a 250 mLtwo-necked round-bottom flask, and the flask was evacuated under vacuumand flushed with dry nitrogen three times. Anhydrous 1,4-dioxane (80 mL)was added, and the mixture was heated to reflux and stirred undernitrogen atmosphere for 24 hours. Afterwards, water was added, and themixture was washed with dichloromethane three times. The organic phasewas combined, dried with MgSO₄, and the solvent was evaporated underreduced pressure. The crude product was purified by silica gel columnchromatography using dichloromethane/hexane (v/v 1:4) as the eluent toaffordN-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-1-amineas a light yellow solid (83% yield).

N-phenyl-N-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)naphthalen-1-amine(2.29 g, 5 mmol), 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole(0.77 g, 2 mmol), K₂CO₃ (2.76 g, 20 mmol), and Pd(PPh₃)₄ (120 mg, 0.1mmol) were added into a 100 mL two-necked round-bottom flask. The flaskwas vacuumed and purged with dry nitrogen three times. Then anhydroustetrahydrofuran (40 mL) and water (10 mL) were added, and the mixturewas heated to reflux and stirred for 24 hours in the absence of light.After cooling to room temperature, water was added, and the mixture wasextracted with dichloromethane three times. The organic phase wascombined and dried with MgSO₄. After removal of the solvent underreduced pressure, the residue was purified by column chromatography onsilica gel using dichloromethane/hexane (v/v 1:3) as the eluent toresult inN,N′-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4,1-phenylene))bis(N-phenylnaphthalen-1-amine)as a dark purple solid (81% yield).

N,N′-((5,6-dinitrobenzo[c][1,2,5]thiadiazole-4,7-diyl)bis(4,1-phenylene))bis(N-phenylnaphthalen-1-amine) (1.22 g, 1.5 mmol) was suspended in a solution of 50mL of acetic acid under vigorous stirring in a 100 mL two-neckedround-bottom flask, and iron power (2.24 g, 40 mmol) was added into themixture. Then the mixture was heated to 80° C. and stirred for 12 hours.After cooling to room temperature, 100 mL of water was added, and themixture was extracted with dichloromethane three times. The organicphase was combined, washed with aqueous NaHCO₃, and dried with MgSO₄.After removal of the solvent under reduced pressure, a dark solid of4,7-bis(4-(naphthalen-1-yl(phenyl)amino)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diaminewas obtained, and it was used without further purification.

4,7-Bis(4-(naphthalen-1-yl(phenyl)amino)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diamine(0.75 g, 1.0 mmol) was dissolved in chloroform (20 mL) and acetic acid(20 mL) in a 100 mL two-necked round-bottom flask under nitrogen. Benzil(0.32 g, 1.5 mmol) was added into this solution. The mixture was heatedto 70° C. and stirred for 12 hours. After cooling to room temperature,100 mL of water was added. The mixture was extracted withdichloromethane three times. The organic phase was combined, washed withaqueous NaHCO₃, and dried with MgSO₄. After removal of the solvent underreduced pressure, the residue was purified by column chromatography onsilica gel using dichloromethane/hexane (v/v 1:3) as the eluent toresult inN,N′-((6,7-diphenyl-[1,2,5]thiadiazolo[3,4-g]quinoxaline-4,9-diyl)bis(4,1-phenylene))bis(N-phenylnaphthalen-1-amine)(NPB-TQ) as a dark blue solid (75% yield).

¹H NMR (400 MHz, CDCl₃, δ, ppm): 8.07 (d, 2H), 7.96-7.89 (m, 6H), 7.83(d, 2H), 7.61 (d, 4H), 7.55-7.46 (m, 6H), 7.42-7.34 (m, 4H), 7.31-7.23(m, 12H), 7.21 (d, 4H), 7.06-6.97 (m, 2H). ¹³C NMR (100 MHz, CDCl₃, δ,ppm): 153.15, 152.58, 148.62, 147.88, 143.22, 138.58, 136.00, 135.34,133.99, 131.39, 130.05, 129.46, 129.24, 128.49, 128.43, 128.16, 127.69,127.34, 126.80, 126.58, 126.43, 126.23, 124.43, 123.13, 122.56, 119.52.HRMS (MALDI) m/z calcd for C₆₄H₄₂N₆S 926.3192, found 926.3170.

Synthesis of TPE-TQ-A

The synthesis of TPE-TQ-A is shown below in Scheme 6.

4,4,5,5-Tetramethyl-2-(4-(1,2,2-triphenylvinyl)phenyl)-1,3,2-dioxaborolane(2.29 g, 5 mmol), 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole(0.77 g, 2 mmol), and Pd(PPh₃)₄ (60 mg, 0.05 mmol) were added into a 100mL two-necked round-bottom flask. The flask was vacuumed and purged withdry nitrogen three times. Then anhydrous tetrahydrofuran (40 mL) wasadded, and the mixture was heated to reflux and stirred for 24 hours inthe absence of light. After cooling to room temperature, water wasadded, and the mixture was extracted with dichloromethane three times.The organic phase was combined and dried with MgSO₄. After removal ofthe solvent under reduced pressure, the residue was purified by columnchromatography on silica gel using dichloromethane/hexane (v/v 1:4) asthe eluent to result in5,6-dinitro-4,7-bis(4-(1,2,2-triphenylvinyl)phenyl)benzo[c][1,2,5]thiadiazoleas a red solid (80% yield).

5,6-Dinitro-4,7-bis(4-(1,2,2-triphenylvinyl)phenyl)benzo[c][1,2,5]thiadiazole(1.33 g, 1.5 mmol) was suspended in a solution of 50 mL of acetic acidunder vigorous stirring in a 100 mL two-necked round-bottom flask, andiron power (2.24 g, 40 mmol) was added into the mixture. Then themixture was heated to 80° C. and stirred for 12 hours. After cooling toroom temperature, 100 mL of water was added. The mixture was extractedwith dichloromethane three times. The organic phase was combined, washedwith aqueous NaHCO₃, and dried with MgSO₄. After removal of the solventunder reduced pressure, a yellow solid(4,7-bis(4-(1,2,2-triphenylvinyl)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diamine)was obtained, and it was used without further purification.

4,7-Bis(4-(1,2,2-triphenylvinyl)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diamine(0.83 g, 1.0 mmol) was dissolved in chloroform (20 mL) and acetic acid(20 mL) in a 100 mL two-necked round-bottom flask under nitrogen. Then1,2-bis(4-ethynylphenyl)ethane-1,2-dione (0.39 g, 1.5 mmol) was addedinto this solution. The mixture was heated to 70° C. and stirred for 12hours. After cooling to room temperature, 100 mL of water was added. Themixture was extracted with dichloromethane three times. The organicphase was combined, washed with aqueous NaHCO₃, and dried with MgSO₄.After removal of the solvent under reduced pressure, the residue waspurified by chromatography on silica gel using dichloromethane/hexane(v/v 1:3) as the eluent to result in6,7-bis(4-ethynylphenyl)-4,9-bis(4-(1,2,2-triphenylvinyl)phenyl)-[1,2,5]thiadiazolo[3,4-g]quinoxaline(TPE-TQ-A) as a dark red solid (65% yield).

¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.78 (d, 4H), 7.54 (d, 4H), 7.47 (d,4H), 7.29 (d, 4H), 7.23-7.05 (m, 30H), 3.22 (s, 2H). ¹³C NMR (100 MHz,CDCl₃, δ, ppm): 153.15, 151.61, 143.93, 143.89, 143.78, 143.71, 141.57,140.72, 138.49, 135.93, 132.65, 132.37, 132.13, 131.62, 131.51, 131.45,131.43, 130.66, 129.85, 129.48, 127.83, 127.76, 127.68, 126.72, 126.59,126.54, 126.49, 123.46, 83.19, 79.25. HRMS (MALDI) m/z calcd forC₇₆H₄₈N₄S 1048.3600, found 1048.3593.

Synthesis of MTPE-BTSe

The synthesis of MTPE-BTSe is shown below in Scheme 7.

4,4′-dimethoxybenzophenone (20 mmol, 4.84 g), 4-bromobenzophenone (24mmol, 6.26 g) and zinc dust (80 mmol, 5.2 g) were added to a 500 mLround-bottom flask. Then the flask was evacuated under vacuum andflushed with dry nitrogen three times. After addition of 250 mL ofanhydrous THF, the mixture was cooled to −10° C., and TiCl₄ (7.7 mL, 70mmol) was added dropwise and stirred for 30 minutes. The mixture washeated to reflux and stirred for 12 hours. After cooling to roomtemperature, the reaction was quenched by 1 M HCl solution, and themixture was extracted with dichloromethane three times. The organicphase was combined, dried with MgSO₄, and the solvent was evaporatedunder reduced pressure. The crude product was purified on silica gelcolumn chromatography using dichloromethane/hexane (v/v 1:5) as theeluent to give4,4′-(2-(4-bromophenyl)-2-phenylethene-1,1-diyl)bis(methoxybenzene) as awhite solid (46% yield).

4,4′-(2-(4-Bromophenyl)-2-phenylethene-1,1-diyl)bis(methoxybenzene)(3.77 g, 8 mmol),[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium (II)(Pd(dppf)Cl₂) (0.22 g, 0.3 mmol), KOAc (3.0 g, 30 mmol), andbis(pinacolato)diboron (3.05 g, 12 mmol) were added into a 100 mLtwo-necked round-bottom flask, and the flask was evacuated under vacuumand flushed with dry nitrogen three times. Anhydrous 1,4-dioxane (40 mL)was added, and the mixture was heated to reflux and stirred undernitrogen atmosphere for 24 hours. Afterwards, water was added, and themixture was washed with dichloromethane three times. The organic phasewas combined, dried with MgSO₄, and the solvent was evaporated underreduced pressure. The crude product was purified by silica gel columnchromatography using dichloromethane/hexane (v/v 1:2) as the eluent toafford2-(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolaneas a white solid (85% yield).

2-(4-(2,2-Bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(2.59 g, 5 mmol), 4,7-dibromo-5,6-dinitrobenzo[c][1,2,5]thiadiazole(0.77 g, 2 mmol), tetrakis(triphenylphosphine)palladium (0) (Pd(PPh₃)₄)(60 mg, 0.05 mmol), and K₂CO₃ (2.07 g, 15 mmol) were added into a 100 mLtwo-necked round-bottom flask. The flask was vacuumed and purged withdry nitrogen three times. Then anhydrous tetrahydrofuran (40 mL) andwater (10 mL) were added and the mixture was heated to reflux andstirred under nitrogen atmosphere for 24 hours in the absence of light.Afterwards, water was added, and the mixture was extracted withdichloromethane. The organic phase was combined and dried with MgSO₄.After removal of the solvent under reduced pressure, the residue waspurified by silica gel column chromatography usingdichloromethane/hexane (v/v 1:2) as the eluent to result in4,7-bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-5,6-dinitrobenzo[c][1,2,5]thiadiazoleas an orange solid (79% yield).

In a 250 mL two-necked round-bottom flask,4,7-Bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)-5,6-dinitrobenzo[c][1,2,5]thiadiazole(1.5 g, 1.5 mmol) was suspended in a solution of 80 mL of acetic acidunder vigorous stirring, and iron power (2.24 g, 40 mmol) was added tothe mixture. The mixture was heated to 80° C. and stirred for 24 hours.After cooling to room temperature, 150 mL of water was added, and themixture was extracted with dichloromethane three times. The organicphase was combined, washed with aqueous NaHCO₃, and dried with MgSO₄.After removal of the solvent under reduced pressure, a dark orange solid(4,7-bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diamine)was obtained, and it was used without further purification.

In a 100 mL two-necked round-bottom flask under nitrogen.4,7-Bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)benzo[c][1,2,5]thiadiazole-5,6-diamine(0.95 g, 1 mmol) was dissolved in anhydrous tetrahydrofuran (40 mL).Selenium dioxide (56 mg, 2.5 mmol) was added into this solution, and themixture was stirred at room temperature for 18 hours. Most of thetetrahydrofuran was evaporated in vacuum and the residue was purified byneutral aluminum oxide column chromatography usingdichloromethane/hexane (v/v 1:2) as the eluent to give4,7-bis(4-(2,2-bis(4-methoxyphenyl)-1-phenylvinyl)phenyl)[1,2,5]selenadiazolo[3,4-f]benzo[c][1,2,5]-thiadiazole(MTPE-BTSe) as a green-yellow solid (2.19 g, 62% yield).

¹H NMR (400 MHz, CDCl₃, δ, ppm): 7.97 (d, 2H), 7.33-7.22 (m, 4H),7.21-7.07 (m, 14H), 7.02-6.92 (m, 6H), 6.73-6.62 (m, 8H), 3.75 (t, 12H).HRMS (MALDI) m/z calcd for C₆₂H₄₆O₄N₄SSe 1022.2405, found 1022.2460.

Synthesis of DCDPP-2TPA

The synthesis of DCDPP-2TPA is shown below in Scheme 8.

1,2-bis(4′-(diphenylamino)-[1,1′-biphenyl]-4-yl) ethane-1,2-dione (600mg, 0.86 mmol), 2,3-diaminomaleonitrile (112 mg, 1.03 mmol), and 40 mLof acetic acid were added into a 100 mL round bottom flask. The reactionwas allowed to stir and reflux at 130° C. for 8 hours. After that, themixture was cooled to room temperature and poured into ice water,followed by extraction with dichloromethane. The collected organic phasewas washed with water several times and then dried over anhydrousNa₂SO₄. After filtration and vacuum distillation, the crude product waspurified by a silica gel column with dichloromethane/hexane (1:2 byvolume) as eluent. A red powder of5,6-bis(4′-(diphenylamino)-[1,1′-biphenyl]-4-yl)pyrazine-2,3-dicarbonitrilewas obtained in yield of 75.5%.

¹H NMR (400 MHz, CD₂Cl₂), δ (ppm): 7.68 (d, 4H), 7.61 (d, 4H), 7.52 (d,4H), 7.30 (t, 8H), 7.12 (16H). ¹³C NMR (100 MHz, CD₂Cl₂), δ (ppm):154.9, 147.4, 143.2, 133.6, 132.6, 130.4, 129.4, 127.7, 126.6, 124.8,123.4, 123.1, 113.5. HRMS (MALDI-TOF): m/z 768.2999 [M⁺, calcd for768.3001].

Synthesis of DCDPP-2TPA4M

The synthesis of DCDPP-2TPA4M is shown below in Scheme 9.

The synthetic method is similar to that of DCDPP-2TPA.1,2-bis(4′-(bis(4-methoxyphenyl)amino)-[1,1′-biphenyl]-4-yl)ethane-1,2-dione(500 mg, 0.61 mmol), 2,3-diaminomaleonitrile (80 mg, 0.73 mmol), and 30mL of acetic acid were added into a 50 mL round bottom flask. Thereaction was allowed to stir and reflux at 130° C. for 8 hours. Afterthat, the mixture was cooled to room temperature and poured into icewater, followed by extraction with dichloromethane. The collectedorganic phase was washed with water several times and then dried overanhydrous Na₂SO₄. After filtration and vacuum distillation, the crudeproduct was purified by a silica gel column with dichloromethane/hexane(1:1 by volume) as eluent, followed by recrystallizing fromdichloromethane/acetonitrile. A orange-red powder of5,6-bis(4′-(bis(4-methoxyphenyl)amino)-[1,1′-biphenyl]-4-yl)pyrazine-2,3-dicarbonitrilewas obtained in yield of 70%.

¹H NMR (400 MHz, CDCl₃), δ (ppm): 7.64 (d, 4H), 7.57 (d, 4H), 7.42 (d,4H), 7.10 (d, 8H), 6.99 (d, 4H), 6.87 (d, 8H), 3.82 (s, 12H). ¹³C NMR(100 MHz, CDCl₃), δ (ppm): 156.2, 154.8, 149.2, 143.5, 140.4, 133.1,130.5, 130.3, 129.2, 127.5, 126.9, 126.5, 120.1, 114.8, 113.4, 55.5.HRMS (MALDI-TOF): m/z 888.3456 [M⁺, calcd for 888.3424].

Synthesis of DCDP-2TPA

The synthesis of DCDP-2TPA is shown below in Scheme 10.

1,2-bis(4-(diphenylamino)phenyl) ethane-1,2-dione (245 mg, 0.45 mmol),5,6-diaminopyrazine-2,3-dicarbonitrile (100 mg, 0.63 mol), trace amountof p-toluenesulfonic acid, and 30 mL of toluene were added into a 50 mLround bottom flask. The reaction was allowed to stir and reflux at 120°C. for 4 days. After reaction, the toluene was removed by vacuumdistillation, and the crude product was extracted by dichloromethane andwashed with water three times. The collected organic was then dried overanhydrous Na₂SO₄. After filtration and vacuum distillation, the crudeproduct was purified by a silica gel column with dichloromethane/hexane(1:1 by volume) as eluent. A dark powder of6,7-bis(4-(diphenylamino)phenyl)pyrazino[2,3-b]pyrazine-2,3-dicarbonitrilewas obtained in yield of 59.7%.

¹H NMR (400 MHz, CD₂Cl₂), δ (ppm): 7.73 (d, 4H), 7.37 (t, 8H), 7.20 (m,12H), 6.95 (d, 4H). ¹³C NMR (100 MHz, CD₂Cl₂), δ (ppm): 161.1, 151.7,146.1, 144.1, 131.8, 129.7, 128.3, 126.3, 125.1, 119.2, 113.4. HRMS(MALDI-TOF): m/z 668.2467 [M⁺, calcd for 668.2437].

Synthesis of DCDP-2TPA4M

The synthesis of DCDP-2TPA4M is shown below in Scheme 11.

The synthetic method is similar to that of DCDP-2TPA.1,2-bis(4′-(bis(4-methoxyphenyl)amino)-[1,1′-biphenyl]-4-yl)ethane-1,2-dione(500 mg, 0.61 mmol), 5,6-diaminopyrazine-2,3-dicarbonitrile (108 mg,0.675 mmol), trace amount of p-toluenesulfonic acid, and 30 mL oftoluene were added into a 50 mL round bottom flask. The reaction wasallowed to stir and reflux at 120° C. for 4 days. After reaction, thetoluene was removed by vacuum distillation, and the crude product wasextracted by dichloromethane and washed with water three times. Thecollected organic was then dried over anhydrous Na₂SO₄. After filtrationand vacuum distillation, the crude product was purified by a silica gelcolumn with dichloromethane/hexane (1:1 by volume) as eluent. A darkgreen powder of6,7-bis(4′-(bis(4-methoxyphenyl)amino)-[1,1′-biphenyl]-4-yl)pyrazino[2,3-b]pyrazine-2,3-dicarbonitrilewas obtained in yield of 46.9%.

¹H NMR (400 MHz, CDCl₃), δ (ppm): 7.89 (d, 4H), 7.63 (d, 4H), 7.49 (d,4H), 7.13 (d, 8H), 7.00 (d, 4H), 6.89 (d, 8H), 3.83 (s, 12H). ¹³C NMR(100 MHz, CDCl₃), δ (ppm): 161.8, 156.3, 149.4, 143.7, 144.4, 140.3,134.0, 132.8, 131.2, 130.1, 127.6, 127.0, 126.3, 120.0, 114.8, 112.8,55.5. HRMS (MALDI-TOF): m/z 940.3503 [M⁺, calcd for 940.3486].

Synthesis and Characterization of DTE-TPECM

The synthesis of DTE-TPECM is shown below in Scheme 12.

1-(4-(1,2-Diphenyl-2-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)vinyl)phenyl)ethan-1-one(1.0 g, 2 mmol),3,3′-(perfluorocyclopent-1-ene-1,2-diyl)bis(5-bromo-2-methylthiophene)(0.47 g, 0.9 mmol), and Pd(PPh₃)₄ (0.12 g, 0.1 mmol) were added into a100 mL two-necked round-bottom flask. The flask was vacuumed and purgedwith dry nitrogen three times. Then THF (30 mL) and aqueous K₂CO₃solution (2 M, 10 mL) were added, and the mixture was heated to refluxand stirred overnight. Water was added and the mixture was extractedwith dichloromethane three times. The organic phase was combined anddried with MgSO₄. After the removal of the solvent under reducedpressure, the crude product was purified by column chromatography onsilica gel using dichloromethane/hexane (v/v 1:1) as the eluent toafford1,1′-(((((perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-4,2-diyl))bis(4,1-phenylene))bis(1,2-diphenylethene-2,1-diyl))bis(4,1-phenylene))bis(ethan-1-one)as a yellow solid (76% yield).

1,1′-(((((Perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-4,2-diyl))bis(4,1-phenylene))bis(1,2-diphenylethene-2,1-diyl))bis(4,1-phenylene))bis(ethan-1-one)(0.56 g, 0.5 mmol), malononitrile (0.1 g, 1.5 mmol), and ammoniumacetate (0.12 g, 1.5 mmol) were added into a 100 mL two-neckedround-bottom flask. The flask was vacuumed and purged with dry nitrogenthree times. Then anhydrous toluene (40 mL) and acetic acid (1 mL) wereadded, and the mixture was heated to reflux and stirred for 4 hours.After cooling down to room temperature, water was added, and the mixturewas extracted with dichloromethane. The organic phase was combined anddried with MgSO₄. After the removal of the solvent under reducedpressure, the crude product was purified by column chromatography onsilica gel using dichloromethane/hexane (v/v 2:1) as the eluent toafford2,2′-((((((perfluorocyclopent-1-ene-1,2-diyl)bis(5-methylthiophene-4,2-diyl))bis(4,1-phenylene))bis(1,2-diphenylethene-2,1-diyl))bis(4,1-phenylene))bis(ethan-1-yl-1-ylidene))dimalononitrile(ROpen-DTE-TPECM) as a yellow solid (78% yield).

¹H NMR (400 MHz, CDCl₃, 25° C.) δ (ppm): 7.32-7.20 (m, 8H), 7.18-7.14(m, 2H), 7.18-6.92 (m, 28H), 2.48 (d, 6H), 1.85 (d, 6H). HRMS(MALDI-TOF, m/z): [M]⁺ calcd for C₇₇H₅₀N₄S₂F₆, 1208.3381; found,1208.3357.

ROpen-DTE-TPECM (open-form compound) exhibits AIE characteristics.RClosed-DTE-TPECM (closed-form compound) does not show any luminescencein soluble state or in aggregate state.

The open-form compound exhibits AIE characteristics and can be used forin vivo fluorescence imaging. There is no fluorescence for theclosed-form compound, but it shows strong absorption in the NIR region.As such, the closed-form compound may be used as a photoacoustic agent.Thus, the present subject matter is a light-driven transformable agentfor in vivo fluorescence and a photoacoustic agent, as well asphotosensitizer for photodynamic therapy.

The ¹H NMR spectrum of ROpen-DTE-TPECM is shown in FIG. 79, and the HRMSof ROpen-DTE-TPECM is shown in FIG. 80. The PL spectra are shown inFIGS. 81, 82, and 84. DLS profiles and SEM images are shown in FIG. 83.PA spectra and amplitudes are shown in FIG. 85. The PL excitationmapping and fluorescence decay are shown in FIG. 86.

Representative PA images of subcutaneous tumor from a living mouse afterintravenous administration of RClosed NPs (800 μM based onRClosed-DTE-TPECM, 100 μL) at designated time intervals are shown inFIG. 87a . A plot of PA intensity at 700 nm in tumor versus timepost-injection of RClosed NPs are shown in FIG. 87b , wherein data arepresented as mean±standard deviation (SD) (n=3 mice). Representativebrightfield images of RClosed NPs-treated tumor-bearing mice before andafter surgery as well as representative fluorescence images of mice withcomplete surgical resection of tumors, followed by 610 nm red lightirradiation at the operative incision site for 5 min are shown in FIG.87c . FL: fluorescence; IT: irradiation time. In FIG. 87d , H&E stainedtissues at the operative incision site in (c) indicate no residualtumors left behind. In FIG. 87e , representative fluorescence images ofRClosed NPs-treated mice with residual tumors post-surgery are shown.The operative incision site was irradiated by 610 nm red light fordifferent time points. The red dashed circles in (c) and (e) indicatethe tumor/operative incision site. The red arrow shows the residualtumors with a diameter below 1 mm. In FIG. 87f , H&E stained tissues atthe operative incision site in (e) confirm the existence of residualtumors. In FIG. 87g , average fluorescence intensity of residual tumorsand surrounding normal tissues in (e) (n=5 mice). ** represents P<0.01,in comparison between residual tumor and normal tissue. Time-dependentbioluminescence imaging of residual tumors from mice in different groupsare shown in FIG. 87h . The tumors were debulked on day 0. The 4T1cancer cells express luciferase, permitting bioluminescence imaging. Theblack arrows indicate the residual tumor. DS: debulking surgery. In FIG.87i , quantitative analysis of bioluminescence intensities of residualtumors from mice with various treatments as indicated. ** in (i)represent P<0.01, in comparison between “DS+NPs+Light” cohort and othergroups.

With the information contained herein, various departures from precisedescriptions of the present subject matter will be readily apparent tothose skilled in the art to which the present subject matter pertains,without departing from the spirit and the scope of the below claims. Thepresent subject matter is not considered limited in scope to theprocedures, properties, or components defined, since the preferredembodiments and other descriptions are intended only to be illustrativeof particular aspects of the presently provided subject matter. Indeed,various modifications of the described modes for carrying out thepresent subject matter which are obvious to those skilled in chemistry,biochemistry, or related fields are intended to be within the scope ofthe following claims.

1. A compound comprising a donor and an acceptor, wherein each acceptoris independently selected from the group consisting of:

wherein each D and D′ is the donor and is independently selected fromthe group consisting of:

wherein each X and X′ is independently selected from the groupconsisting of: O, S, Se, and Te; wherein each R, R′, R″, R′″, and R″″ isindependently selected from the group consisting of: F, H, alkyl,unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, carboxyl group, amino group, sulfonic group, alkylthio,alkoxy group, and

and wherein when any of R, and R′, R″, R′″, and R″″ is a terminalfunctional group, then each terminal functional group R, R′, R″, R′″,and R″″ is independently selected from the group consisting of N₃, NCS,SH, NH₂, COOH, alkyne, N-Hydroxysuccinimide ester, maleimide, hydrazide,nitrone group, CHO, —OH, halide, and charged ionic group; wherein eachR₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ may be substituted orunsubstituted and each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isindependently selected from the group consisting of H, alkyl,unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, C_(m)H_(2m+1), C₁₀H₇, C₁₂H₉, OC₆H₅, OC₁₀H₇, OC₁₂H₉,C_(m)H_(2m)COOH, C_(m)H_(2m)NCS, C_(m)H_(2m)N₃, C_(m)H_(2m)NH₂,C_(m)H_(2m)SO₃, C_(m)H_(2m)Cl, C_(m)H_(2m)Br, C_(m)H_(2m)I,

wherein n=0-2; and wherein m=0 to 20; provided that when the acceptor is

the donor is

and X is S, then at least one of R, R′, and R″ is not H.
 2. The compoundof claim 1, wherein at least one donor and at least one acceptor arearranged in an order selected from the group consisting of:Donor-Acceptor, Donor-Acceptor-Donor, Acceptor-Donor-Acceptor,Donor-Donor-Acceptor-Donor-Donor,Acceptor-Acceptor-Donor-Acceptor-Acceptor,Donor-Acceptor-Donor-Acceptor-Donor, andAcceptor-Donor-Acceptor-Donor-Acceptor.
 3. The compound of claim 1,wherein the compound comprises a structure of:

wherein each X′ is independently selected from the group consisting of:S, Se, and Te; wherein each R, R′, and R″ is independently selected fromthe group consisting of: F, H, alkyl, unsaturated alkyl, heteroalkyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, aminogroup, sulfonic group, alkylthio, and alkoxy group; and wherein when anyof R, R′, and R″ is a terminal functional group, then each terminalfunctional group R, R′, and R″ is independently selected from the groupconsisting of N₃, NCS, SH, NH₂, COOH, alkyne, N-Hydroxysuccinimideester, maleimide, hydrazide, nitrone group, —CHO, —OH, halide, andcharged ionic group.
 4. The compound of claim 3, wherein the compoundcomprises a structure of:


5. The compound of claim 4, wherein the compound is selected from thegroup consisting of:


6. The compound of claim 1, wherein the compound exhibitsaggregation-induced emission (AIE).
 7. A probe comprising the compoundof claim 1, wherein the probe is a far red/near-infrared (FR/NIR)fluorescent probe.
 8. The probe of claim 7, wherein the compound isfunctionalized with special targeted groups to image biological species.9. The probe of claim 7, wherein the compound is in a PEG matrix is in aform of nanoparticles.
 10. The probe of claim 9, wherein thenanoparticles can be incubated with cells and used to image cellularcytoplasms.
 11. The compound of claim 1, wherein the compound is apyrazine-based red/near-infrared AIEgen wherein each acceptor isindependently selected from the group consisting of:

wherein each D and D′ is the donor and is independently selected fromthe group consisting of:

wherein n=0-2; wherein each X is independently O or S; wherein each R,R′, and R″ is independently selected from the group consisting of: F, H,alkyl, unsaturated alkyl, heteroalkyl, heterocycloalkyl, aryl,heteroaryl, carboxyl group, amino group, alkylthio, sulfonic group, andalkoxy group; and wherein when any of R, R′, and R″ is a terminalfunctional group, then each terminal functional group R, R′, and R″ isindependently selected from the group consisting of methoxyl, tertiarybutyl, N₃, NCS, SH, NH₂, COOH, alkyne, N-Hydroxysuccinimide ester,maleimide, hydrazide, nitrone group, —CHO, —OH, halide, and chargedionic group.
 12. The compound of claim 11, wherein each D and D′ is

wherein n=0-2; wherein each R, R′, and R″ is independently selected fromthe group consisting of: F, H, alkyl, unsaturated alkyl, heteroalkyl,heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group,alkylthio, sulfonic group, and alkoxy group; and wherein when any ofeach R, R′, and R″ is a terminal functional group, then each terminalfunctional group R, R′, and R″ is independently selected from the groupconsisting of methoxyl, tertiary butyl, N₃, NCS, SH, NH₂, COOH, alkyne,N-Hydroxysuccinimide ester, maleimide, hydrazide, nitrone group, —CHO,—OH, halide, and charged ionic group.
 13. The compound of claim 11,wherein the compound comprises a structure of:

wherein n=1-3; wherein each R, R′, R″, and R′″ is independently selectedfrom the group consisting of: F, H, alkyl, unsaturated alkyl,heteroalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, aminogroup, alkylthio, sulfonic group, and alkoxy group; and wherein wheneach R, R′, and R″ is a terminal functional group, then each R, R′, andR″ is independently selected from the group consisting of methoxyl,tertiary butyl, N₃, NCS, SH, NH₂, COOH, alkyne, N-Hydroxysuccinimideester, maleimide, hydrazide, nitrone group, —CHO, —OH, halide, andcharged ionic group.
 14. The compound of claim 11, wherein the compoundis


15. The compound of claim 11, wherein the compound exhibitsaggregation-induced emission (AIE).
 16. A probe comprising the compoundof claim 11, wherein the probe is a red/near-infrared fluorescent probe.17. The probe of claim 16, wherein the compound is functionalized withspecial targeted groups to image biological species.
 18. The probe ofclaim 16, wherein the compound is fabricated in PEG/BSA and anyamphiphilic molecule matrix, wherein the probe works in a form ofnanoparticles.
 19. The probe of claim 18, wherein the nanoparticles areincubated with cells or tissue and used for imaging cellular cytoplasmsor tissue.
 20. The probe of claim 18, wherein the nanoparticles areinjected into a blood vessel and used for blood vessel imaging. 21.(canceled)
 22. (canceled)
 23. (canceled)
 24. The compound of claim 1,wherein the compound comprises a backbone structure selected from thegroup consisting of:

wherein each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ may be substitutedor unsubstituted and each R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈ and R₉ isindependently selected from the group consisting of H, alkyl,unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, C_(m)H_(2m+1), C₁₀H₇, C₁₂H₉, OC₆H₅, OC₁₀H₇, OC₁₂H₉,C_(m)H_(2m)COOH, C_(m)H_(2m)NCS, C_(m)H_(2m)N₃, C_(m)H_(2m)NH₂,C_(m)H_(2m)SO₃, C_(m)H_(2m)Cl, C_(m)H_(2m)Br, C_(m)H_(2m)I,

wherein m=0 to 20; and wherein X is independently selected from S andSe.
 25. The compound of claim 24, wherein the compound is TTS having astructure of:


26. A probe comprising the compound of claim 24, wherein the probe isused for brain vascular imaging, sentinel lymph node mapping, and tumorimaging.